Technology for preparation of macromolecular microspheres

ABSTRACT

Microspheres are produced by contacting an aqueous solution of a protein or other macromolecule with an organic solvent and a counterion, and chilling the solution. The microspheres are useful for preparing pharmaceuticals of defined dimensions.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/148,543, filed Jan. 6, 2014, which is a continuation of U.S.application Ser. No. 13/289,644, filed Nov. 4, 2011, which is acontinuation of U.S. application Ser. No. 12/633,773, filed Dec. 8,2009, which is a continuation of U.S. application Ser. No. 11/657,812,filed Jan. 24, 2007, which claims priority to U.S. ProvisionalApplication No. 60/762,002, filed Jan. 24, 2006, all of which are herebyincorporated by reference in its entirety.

This application also is related to International PCT applicationapplication Serial No. (Attorney Docket No. 21865-004WO1/6504PC, filedJan. 24, 2007). This application also is related to published U.S.applications Serial Nos. US20050004020 A1 and US20050112751 A1. Each ofthese applications is incorporated by reference in its entirety.

BACKGROUND

The administration of proteins to animals, including humans, innutritional supplements or as therapeutics has been known for some time.Proteins for therapeutic or nutritional administration generally areavailable either as (1) concentrates or powders that are administereddirectly or are reconstituted in a liquid of choice prior to use; or (2)liquid formulations.

The preparation and delivery of therapeutic proteins of interest inpowder or particle form is an area of concentrated research anddevelopment activity in the pharmaceutical industry. For therapeuticefficacy, it is desirable to have a uniform formulation. For example,for pulmonary administration, the protein ideally is prepared in theform of discrete microspheres, which are solid or semi-solid particleshaving a diameter of between 0.5 and 5.0 microns. It also is desirablefor the particles to have a protein content that is as high as possibleand that maintains its activity for concentrated delivery andtherapeutic efficacy.

Previous methods of producing protein microparticles or nanoparticleshave involved complex steps, such as blending with organic polymersand/or forming a lattice array with polymers; spray drying, sprayfreeze-drying or supercritical fluid antisolvent techniques that usespecialized and complex equipment; or lyophilization followed bypulverization or milling that often results in non-uniform particlesthat must further be sorted. Often previous methods of producing solidprotein formulations involve processing steps, such as heating, thatdenature the protein and compromise its activity. In addition, somemethods do not provide high recovery from solution into the solidformulation.

Accordingly, there is a need for a method for producing protein andother macromolecular microparticles that does not require complex orspecialized equipment and that produces uniform-sized microparticles fordelivery. There further is a need for a method of producingmicroparticles that contain high concentrations of the protein ormacromolecule relative to other components, that are stable and maintaintheir activity for long periods of time when stored at ambienttemperature and that do not contain a significant amount of denaturedprotein. There also is a need for a method of producing microparticlesof proteins and other macromolecules wherein substantially all of theprotein or macromolecule in the starting material is recovered in themicroparticle formulation, with minimal loss. There also is a need formicroparticles of proteins or other macromolecules containing theseproperties for administration, for example, as a therapeutic ornutritional supplement.

SUMMARY

Provided herein are methods for producing protein and othermacromolecular microparticles that do not require complex or specializedequipment and that produces uniform-sized microparticles for delivery;methods for producing microparticles that contain high concentrations ofprotein or macromolecule relative to other components, that are stableand maintain their activity for long periods of time when stored atambient temperature and that do not contain a significant amount ofdenatured protein. Also provide are methods for producing microparticlesof proteins and other macromolecules where substantially all of theprotein or macromolecule in the starting material is recovered in themicroparticle formulation, with minimal loss. Also provided aremicroparticles of proteins and other macromolecules containing theseproperties for administration, for example, as a therapeutic ornutritional supplement.

The methods of making a protein-based composition, the protein-basedcompositions themselves, combinations, articles of manufacture providedbelow are characterized by a variety of component ingredients, steps ofpreparation, and biophysical, physical, biochemical and chemicalparameters. As would be apparent to one of skill in the art, thecompositions and methods provided herein include any and allpermutations and combinations of the ingredients, steps and/orparameters described below.

Provided herein are methods of making a protein-based composition. Themethod provided herein can be used to make compositions from othermacromolecules besides proteins, including DNA, RNA, PNA, lipids,oligosaccharides and combinations thereof.

The methods provided herein can include the steps of:

a) adding a counterion to a solution containing the protein in anaqueous solvent;

b) adding an organic solvent to the solution; and

c) gradually cooling the solution to a temperature below about 25° C.,whereby a composition containing microparticles comprising the proteinis formed, wherein steps a), b) and c) are performed simultaneously,sequentially, intermittently, or in any order.

In one embodiment, the steps are performed sequentially a), b) and thenc). In another embodiment, the method of making a protein-basedcomposition includes performing steps a) and b) simultaneously orsequentially in any order, followed by step c).

The resulting microparticles can be obtained by precipitation, by phaseseparation or by colloid formation. In some aspects, the methodsprovided herein further comprise separating the microparticles from thesolution to remove components other than the microparticles. Thisseparation step can be performed following the above-mentioned step c).The separation can be effected by, for example, sedimentation,filtration and/or freeze-drying.

The methods provided herein include the addition of an organic solventto an aqueous solvent containing the protein. In certain embodiments,the organic solvent in miscible or partially miscible with the aqueoussolvent. In further embodiments of the methods provided herein, theorganic solvent is selected from among aliphatic alcohols, aromaticalcohols, chloroform, dimethyl chloride, polyhydric sugar alcohols,aromatic hydrocarbons, aldehydes, ketones, esters, ethers, dioxanes,alkanes, alkenes, conjugated dienes, dichloromethane, acetonitrile,ethyl acetate, polyols, polyimides, polyesters, polyaldehydes andmixtures thereof. For example, where the organic solvent is an aliphaticalcohol, the organic solvent can be isopropanol. The amount of organicsolvent added can vary in the methods provided herein. For example, theamount of organic solvent added can be from about 0.1% or 0.1% to about50% or 50% v/v. In other embodiments, the amount of organic solventadded is from about 1% or 1% to about 30% or 30% v/v, from about 5% or5% to about 30% or 30% v/v, from about 10% or 10% to about 30% or 30%v/v or from about 15% or 15% to about 20% or 20% v/v.

The counterion used in the methods provided herein can be an anioniccompound, a cationic compound and/or a zwitterionic compound. Forexample, when the counterion is an anionic compound, the counterion canbe glycine, sodium citrate, sodium sulfate, zinc sulfate, magnesiumsulfate, potassium sulfate or calcium sulfate. The concentration oforganic solvent added to the solution can vary in the method providedherein. For example, the concentration of counterion added to thesolution can be from about 0.1 mM or 0.1 mM to about 100 mM or 100 mM.In other embodiments, the concentration of counterion added to thesolution is from about 0.2 mM or 0.2 mM to about 50 mM or 50 mM, fromabout 0.3 mM or 0.3 mM to about 30 mM or 30 mM, from about 0.5 mM or 0.5mM to about 20 mM or 20 mM or from about 1 mM or 1 mM to about 10 mM or10 mM. In a particular embodiment, the concentration of counterion addedto the solution is about 5 mM or 5 mM.

In one aspect of the methods provided herein, the pH of the solutionthat contains the protein is at or below the pI of the protein. In someaspects, the pH of the solution is from about 4.0 or 4.0 to about 9.0 or9.0. In other aspects, the pH of the solution is from about 4.5 or 4.5to about 8.0 or 8.0, from about 4.5 or 4.5 to about 6.5 or 6.5, or fromabout 4.5 or 4.5 to about 5.5 or 5.5.

The protein that is used in the methods provided herein to make aprotein-based composition can be selected from among sialidases,sialidase fusion proteins, proteases, protease inhibitors, cytokines,insulin, human growth hormone, calcitonin, recombinant human DNase,interferons and parathyroid hormone. In one embodiment, where theprotein is a protease inhibitor, the protein is human protease inhibitor8 (PI8). In another embodiment, the protein is a sialidase fusionprotein. In some aspects where the protein is a sialidase fusionprotein, the sialidase fusion protein contains a catalytic domain of asialidase and an anchoring domain, wherein the catalytic domain of thesialidase is the only portion of the sialidase in the sialidase fusionprotein. The sialidase can be, for example, an Actinomyces viscosussialidase, a Clostridium perfringens sialidase, an Arthrobacterureafaciens sialidase, a Micromonospora viridifaciens sialidase, a humanNeu2 sialidase or a human Neu4 sialidase.

In one aspect, where the sialidase is an Actinomyces viscosus sialidase,the amino acid sequence of the catalytic domain contains the sequence ofamino acid residues beginning at any of the amino acids from amino acid270 to amino acid 290 and ending at any of the amino acids from aminoacid 665 to amino acid 901 of the sequence of amino acids set forth inSEQ ID NO. 1. For example, in one embodiment, the sequence of thesialidase catalytic domain contains the sequence of amino acid residuesset forth in SEQ ID NO:2. In another embodiment, the sequence of thecatalytic domain comprises the sequence of amino acid residues beginningat amino acid 274 and ending at amino acid 681 of the sequence of aminoacids set forth in SEQ ID NO. 1. In a different embodiment, the sequenceof the catalytic domain comprises the sequence of amino acid residuesbeginning at amino acid 274 and ending at amino acid 666 of the sequenceof amino acids set forth in SEQ ID NO. 1. In another embodiment, thesequence of the catalytic domain comprises the sequence of amino acidsbeginning at amino acid 290 and ending at amino acid 681 of the sequenceof amino acids set forth in SEQ ID NO. 1.

In one aspect, where the protein that is used in the methods providedherein to make a protein-based composition is a sialidase fusion proteinthat contains an anchoring domain, the anchoring domain is aglycosaminoglycan (GAG)-binding domain. In a further aspect, theGAG-binding domain is selected from among the GAG-binding domain ofhuman platelet factor 4, the GAG-binding domain of human interleukin 8,the GAG-binding domain of human antithrombin III, the GAG-binding domainof human apoprotein E, the GAG-binding domain of human angio-associatedmigratory protein and the GAG-binding domain of human amphiregulin. Inparticular embodiments, the amino acid sequence of the GAG-bindingdomain contains the sequence of amino acid residues set forth in SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

In some aspects where the protein that is used in the methods providedherein to make a protein-based composition is a sialidase fusionprotein, the amino acid sequence of the sialidase fusion proteincontains the sequence of amino acid residues set forth in SEQ ID NO:9,the sequence of amino acid residues set forth in SEQ ID NO:10, thesequence of amino acid residues set forth in SEQ ID NO:11, the sequenceof amino acid residues set forth in SEQ ID NO:12, the sequence of aminoacid residues set forth in SEQ ID NO:13, the sequence of amino acidresidues set forth in SEQ ID NO:14, or the sequence of amino acidresidues set forth in SEQ ID NO:17.

In one aspect, the amount of protein in the microparticles produced bythe methods provided herein, relative to the total amount of protein inthe solution of step a) is about 80% or 80% to greater than about 99% or99%. In another aspect, the resulting microparticle composition producedby the methods provided herein can further comprise acid-resistantcoating agents, protease-resistant coating agents, enteric coatingagents, bulking agents, excipients, inactive ingredients, stabilityenhancers, taste and/or odor modifiers or masking agents, vitamins,therapeutic agents, anti-oxidants, immuno-modulators, trans-membranetransport modifiers, anti-caking agents, chitosans or flowabilityenhancers.

The solution and/or the resulting composition of the methods providedherein can, in one aspect, further comprise an active agent. In someembodiments, the active agent is selected from among antidiabetics,anticonvulsants, analgesics, antiparkinsons, anti-inflammatories,calcium antagonists, anesthetics, antimicrobials, antimalarials,antiparasitics, antihypertensives, antihistamines, antipyretics,alpha-adrenergic agonists, alpha-blockers, biocides, bactericides,bronchial dilators, beta-adrenergic blocking drugs, contraceptives,cardiovascular drugs, calcium channel inhibitors, depressants,diagnostics, diuretics, electrolytes, enzymes, hypnotics, hormones,hypoglycemics, hyperglycemics, muscle contractants, muscle relaxants,neoplastics, glycoproteins, nucleoproteins, lipoproteins, ophthalmics,psychic energizers, sedatives, steroids, sympathomimetics,parasympathomimetics, tranquilizers, urinary tract drugs, vaccines,vaginal drugs, vitamins, minerals, nonsteroidal anti-inflammatory drugs,angiotensin converting enzymes, polynucleotides, polypeptides andpolysaccharides. In another embodiment, the active agent is anutritional supplement.

The methods provide herein involve gradually cooling the solution to atemperature below about 25° C. In one embodiment, the temperature isbetween about 4° C. to about −45° C. In another embodiment, thetemperature is between about 2° C. to about −20° C. In a furtherembodiment, the temperature is between about 2° C. to about −15° C. Inanother embodiment, the temperature is between about 0° C. or 0° C. toabout −2° C. or −2° C. to from about −15° C. or −15° C. to about −20° C.or −20° C. The gradual cooling can be performed at a variety of rates.For example, cooling can be effected at a rate of from about 0.01°C./min or 0.01° C./min to about 20° C./min or 20° C./min. In otherembodiments, the gradual cooling is at a rate of from about or at 0.05°C./min or about or at 0.1° C./min to about or at 10° C./min or about orat 15° C./min, about or at 0.2° C./min to about or at 5° C./min, orabout or at 0.5° C./min to about or at 2° C./min. In a particularembodiment, the gradual cooling is performed at a rate of about or at 1°C./min.

In one aspect, the size of the microparticles produced by the methodsprovided herein is from about 0.001 μm or 0.001 μm to about 50 μm or 50μm. In other embodiments, the size of the microparticles is from about0.3 μm or 0.3 μm to about 30 μm or 30 μm, from about 0.5 μm or 0.5 μm toabout 10 μm or 10 μm, from about 0.5 μm or 0.5 μm to about 5.0 μm or 5.0μm, from about 1.0 μm or 1.0 μm to about 5.0 μm or 5.0 μm or from about1.0 μm to about 2.0, 3.0, 4.0 or 5.0 μm.

In some aspects of the methods provided herein, the resultingprotein-based composition has a shelf life of from about one week toabout 1 month, from about 1 month to about six months, from about sixmonths to about one year, from about 1 year to about 2 years, or fromabout 2 years to about 5 years at a temperature of about 55° C., 50° C.,45° C., 44° C., 42° C., 40° C., 39° C., 38° C., 37° C. or below. 54. Inanother aspect, the moisture content of the microparticles is adjustedwhereby at least about 90% of the activity of the protein is retainedafter storage for about six months to about 1 year at a temperature ofabout 25° C. In another aspect, the moisture content of themicroparticles is adjusted whereby at least about 90% of themicroparticles are not aggregated after storage for about six months toabout 1 year at a temperature of about 25° C.

In a certain aspect of the methods provided herein, the protein is afusion protein containing a sialidase catalytic domain and an anchoringdomain, wherein the sialidase catalytic domain is the only portion ofthe sialidase in the fusion protein, the organic solvent is added in anamount of about 5% or 5% to about 20% or 20% v/v, the counterion isadded in an amount of about 1 mM or 1 mM to about 5 mM or 5 mM, and thepH of the solution is adjusted to about 4.5 or 4.5 to about 5.5 or 5.5.In one embodiment of this aspect, the sialidase catalytic domain is fromActinomyces viscosus and the anchoring domain is the GAG-binding domainfrom human amphiregulin. Further still, the pH is about 5.0 and/or thecounterion is selected from among glycine, sodium citrate, sodiumsulfate, zinc sulfate, magnesium sulfate, potassium sulfate or calciumsulfate. In another embodiment of this aspect, the organic solvent isisopropanol. In one embodiment of this aspect, the resulting compositioncontains the microparticles containing the protein as the only activeingredient (i.e. consists essentially of). In another embodiment, themethod includes separating the microparticles from the solution toremove components other than the microparticles, such as bysedimentation, filtration and/or freeze-drying. In some embodiments ofthis aspect, the moisture content of the microparticles is from about 6%to about 12%, or from about 7% to about 10.5%.

Provided herein are compositions containing microparticles of asialidase or a sialidase fusion protein. Where the protein is asialidase fusion protein, the sialidase fusion protein can comprise acatalytic domain of a sialidase and an anchoring domain. In someaspects, the sialidase in the composition is an Actinomyces viscosussialidase, a Clostridium perfringens sialidase, an Arthrobacterureafaciens sialidase, a Micromonospora viridifaciens sialidase, a humanNeu2 sialidase, or a human Neu4 sialidase.

In one aspect, where the sialidase of the composition is an Actinomycesviscosus sialidase, the amino acid sequence of the catalytic domaincomprises the sequence of amino acids beginning at any of the amino acidresidues from amino acid 270 to amino acid 290 and ending at any of theamino acid residues from amino acid 665 to amino acid 901 of thesequence of amino acids set forth in SEQ ID NO. 1. For example, thesequence of the catalytic domain can comprise the sequence of aminoacids beginning at amino acid 274 and ending at amino acid 681 of thesequence of amino acids set forth in SEQ ID NO. 1, the sequence of aminoacids beginning at amino acid 290 and ending at amino acid 666 of thesequence of amino acids set forth in SEQ ID NO. 1 or the sequence ofamino acids beginning at amino acid 290 and ending at amino acid 681 ofthe sequence of amino acids set forth in SEQ ID NO. 1. In anotherembodiment, the sequence of the sialidase catalytic domain comprises thesequence of amino acids set forth in SEQ ID NO:2.

In one aspect, where the composition comprising microparticles of asialidase fusion protein, the anchoring domain of the sialidase fusionprotein is a glycosaminoglycan (GAG)-binding domain. In a furtheraspect, the GAG-binding domain is selected from among the GAG-bindingdomain of human platelet factor 4, the GAG-binding domain of humaninterleukin 8, the GAG-binding domain of human antithrombin III, theGAG-binding domain of human apoprotein E, the GAG-binding domain ofhuman angio-associated migratory protein and the GAG-binding domain ofhuman amphiregulin. In particular embodiments, the amino acid sequenceof the GAG-binding domain contains the sequence of amino acid residuesset forth in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7 or SEQ ID NO:8.

The sialidase fusion proteins of the compositions provided herein cancontain, for example, the sequence of amino acid residues set forth inSEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, or SEQ ID NO:17.

The amount of protein in the microparticles of the compositions providedherein can vary. For example, the amount of protein in themicroparticles can be from about 60% to greater than about 99% w/w. Inone embodiment, the amount of protein in the microparticles is fromabout 65% to about 90% w/w. In another embodiment, the amount of proteinin the microparticles is from about 70% to about 85%, 86%, 87%, 88%, 89%or 90% w/w. The amount of protein in the microparticles of thecompositions provided herein also can be from about 90% to about 99%w/w.

The microparticles in the compositions provided herein can furthercontain acid-resistant coating agents, protease-resistant coatingagents, enteric coating agents, bulking agents, excipients, inactiveingredients, stability enhancers, taste and/or odor modifiers or maskingagents, vitamins, therapeutic agents, anti-oxidants, immuno-modulators,trans-membrane transport modifiers, anti-caking agents, chitosans orflowability enhancers.

In one aspect, the compositions provided herein can further contain anactive agent. The active agent can be a nutritional supplement,antidiabetics, anticonvulsants, analgesics, antiparkinsons,anti-inflammatories, calcium antagonists, anesthetics, antimicrobials,antimalarials, antiparasitics, antihypertensives, antihistamines,antipyretics, alpha-adrenergic agonists, alpha-blockers, biocides,bactericides, bronchial dilators, beta-adrenergic blocking drugs,contraceptives, cardiovascular drugs, calcium channel inhibitors,depressants, diagnostics, diuretics, electrolytes, enzymes, hypnotics,hormones, hypoglycemics, hyperglycemics, muscle contractants, musclerelaxants, neoplastics, glycoproteins, nucleoproteins, lipoproteins,ophthalmics, psychic energizers, sedatives, steroids, sympathomimetics,parasympathomimetics, tranquilizers, urinary tract drugs, vaccines,vaginal drugs, vitamins, minerals, nonsteroidal anti-inflammatory drugs,angiotensin converting enzymes, polynucleotides, polypeptides andpolysaccharides.

The compositions provided herein can have a shelf-life of varyinglength. In one aspect, the shelf life is from about one week to about 1month, from about 1 month to about six months, from about six months toabout one year, from about 1 year to about 2 years, or from about 2years to about 5 years at a temperature of about 55° C., 50° C., 45° C.,44° C., 42° C., 40° C., 39° C., 38° C., 37° C. or below. In a certainaspect, the moisture content of the microparticles is adjusted wherebyat least about 90% of the activity of the protein is retained afterstorage for about six months to about 1 year at a temperature of about25° C.

The microparticles in the compositions provided herein can furthercontain a counterion, such as, for example, an anion, a cation, or azwitterion. In certain embodiments, the counterion is selected fromamong glycine, sodium citrate, sodium sulfate, zinc sulfate, magnesiumsulfate, potassium sulfate or calcium sulfate. The amount of counterionin a microparticle can be varied. For example, the amount of counterionin the microparticles can be from about 0.5% or 0.5% to about 5% or 5%w/w, from about 0.5% or 0.5% to about 2% or 2% w/w, or from about 1% or1% to about 2% or 2% w/w.

In some embodiments, the moisture content of the microparticles in thecompositions provided herein is from about 6% or 6% to about 12% or 12%,or from about 7% or 7% to about 10.5% or 10.5%.

The compositions provided herein can be formulated for a variety ofmodes of administration. For example, the compositions can be orallye.g. by ingestion, intravenously, intranasally, parenterally,subcutaneously or intramuscularly administered. The compositions alsocan formulated for pulmonary or ophthalmic administration. In a certainaspect, the composition provided herein is for inhalation.

The compositions provided herein can be formulated as tablets, caplets,gels, vials, pre-filled syringes, inhalers, electrostatic devices andother devices for delivery. The delivery dosage of the compositions canbe from between about 0.5 mg protein per dose to about 100 mg proteinper dose, or about 0.75 mg, 1 mg, 1.5 mg, 2 mg, 3 mg, 5 mg, 10 mg, 15mg, 20 mg, 30 mg, 40 mg, 45 mg, 50 mg, 55 mg or 60 mg protein per dose.The frequency of administration of a dose, for example, for thetreatment or prophylaxis of influenza, can be from three or more times aday, to two times a day, to once a day, to two times a week, to once aweek, to once every two weeks or less frequent than once every twoweeks. For prohylaxis, the administration generally can be of the orderof about once every two weeks or less frequent, such as once every threeweeks or once every four weeks or longer.

The size of the microparticles in the compositions provided herein canvary. For example, the size of the microparticles can be from about0.001 μm or 0.001 μm to about 50 μm or 50 μm. In certain embodiments,the size of the microparticles is from about 0.3 μm or 0.3 μm to about30 μm or 30 μm, from about 0.5 μm or 0.5 μm to about 10 μm or 10 μm,from about 0.5 μm or 0.5 μm to about 5.0 μm or 5.0 μm, from about 1.0 μmor 1.0 μm to about 5.0 μm or 5.0 μm or from about 1.0 μm to about 2.0,3.0, 4.0 or 5.0 μm.

Also provided herein are articles of manufacture that contain acomposition containing microparticles of a sialidase or a sialidasefusion protein, a packaging material for the composition and a labelthat indicates that the composition is for a therapeutic indication. Inone embodiment, the therapeutic indication is influenza. The article ofmanufacture also can contain an inhaler for pulmonary administration ofthe composition. In certain embodiments, the inhaler is a dry powderinhaler, a metered dose inhaler or an electrostatic delivery device.

DETAILED DESCRIPTION A. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the invention(s) belong. All patents, patent applications,published applications and publications, Genbank sequences, websites andother published materials referred to throughout the entire disclosureherein, unless noted otherwise, are incorporated by reference in theirentirety. In the event that there are a plurality of definitions forterms herein, those in this section prevail. Where reference is made toa URL or other such identifier or address, it understood that suchidentifiers can change and particular information on the internet cancome and go, but equivalent information can be found by searching theinternet. Reference thereto evidences the availability and publicdissemination of such information.

As used herein, the term “macromolecule” is used to mean a moleculecomposed of two or more monomeric subunits, or derivatives thereof,which are linked by a bond, or any macromolecule that can form tertiarystructure. A macromolecule can be, for example, a polynucleotide, anucleic acid molecules including DNA, RNA and peptide nucleic acid (PNA)a polypeptide, a protein, a carbohydrate, or a lipid, or derivatives orcombinations thereof, for example, a nucleic acid molecule containing apeptide nucleic acid portion or a glycoprotein, respectively. Themethods, compositions, combinations, kits and articles of manufactureprovided herein, although described with reference to proteins, can beadapted for use with other macromolecules as defined and providedherein.

The term “substantially” or “substantial” as used herein generally meansat least about 60% or 60%, about 70% or 70%, or about or at 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or higher relative to a reference suchas, for example, a nucleic acid or protein sequence or the originalcomposition of an entity. Thus, a composition containing microparticlesseparated from “substantially” all other contaminants and/or ingredientsincluding counterions, salts and solvents from the cocktail solutionmeans that at least about 60% or 60%, about 70% or 70%, or about or at75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher amounts ofcontaminants and/or reagents have been removed from the cocktailsolution in which the microparticles are formed. The term “substantiallyidentical” or “substantially homologous” or similar varies with thecontext as understood by those skilled in the relevant art and generallymeans at least about 60% or 60%, about 70% or 70%, or about or at 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher identity.

The term “consists essentially of” or “consisting essentially of” asused herein refers to an entity from which substantially all othercomponents/ingredients that are not associated with the entity or itsproperties have been removed or separated from the entity. Thus, acomposition “consisting essentially of” microparticles means that allother ingredients such as contaminants and and solvents havesubstantially been removed from the solution/suspension containing themicroparticles.

The term “microparticle” as used herein is interchangeable with“microsphere” and refers to particles in the size range (average length,width or diameter) of about or at 0.001 micron (μm) to about or at 500microns that contain a macromolecule and deliver an agent of interest,such as a drug or nutritional supplement, to a subject. The agent can bethe macromolecule, for example, a protein, nucleic acid, lipid orpolysaccharide, or the macromolecule forming the microparticle can be acarrier for the active agent, such as a drug or a nutritionalsupplement. The microparticles also can contain synthetic macromoleculesincluding polymers, such as polyethylene glycol (PEG), polylactic acid(PLA), polylactic-co-glycolic acid (PLGA), and natural polymers such asalbumin, gelatin, chitosan and dextran. The “microparticles” asdescribed herein can contain and can be made from a particular naturalor synthetic macromolecule alone, or from more than one type of the samenatural or synthetic macromolecule (e.g., more than one type ofprotein), or from combinations of more than one different type ofnatural or synthetic macromolecule (e.g., a protein and a nucleic acidor a protein and a synthetic polymer).

The term “microparticle” as used herein also generally refers to aparticle that is not a solid form of the entire solution from which itis produced, although frozen and/or dried particles of a solutioncontaining macromolecules also are contemplated herein. Rather, themicroparticle as used herein generally is an assembly of a fraction ofthe components of a solution, including salts, counterions, solvents andother ingredients, that is formed by a process including, but notlimited to, precipitation, sedimentation, phase separation and colloidformation.

The term “precipitation” as used herein refers to a process whereby asolute or solutes of interest in a solution, such as the components of amicroparticle, no longer stay in solution and form a phase that isdistinct from the solvent or solvents that were used to form thesolution. Precipitation of a microparticle and controlling the size ofthe precipitated microparticle can be accomplished by a variety of meansincluding, but not limited to, adjusting temperature, ionic strength,pH, dielectric constant, counterion concentration, organic solventconcentration, the addition of polyelectrolytes or polymers,surfactants, detergents, or a combination thereof.

The term “phase separation” as used herein refers to the transformationof a single homogeneous phase, such as a solution, into two or morephases, such as a suspension of a solid particle in a solvent orsolution.

The term “sedimentation” as used herein refers to the motion ofparticles, such as microparticles, which are in a suspension in a liquidor which are formed in a solution in response to an external force suchas gravity, centrifugal force or electric force.

The term “solution” is used interchangeably with “cocktail solution”herein and refers to a homogeneous mixture of two or more ingredients ina single phase, solid, liquid, or gas, where the distinct ingredientsonly are recognizable at the molecular level. The solution can be aliquid in which one or more solutes, such as salts, are dissolved in asolvent, such as water or alcohol, or dissolved in a mixture of misciblesolvents, such as a mixture of water and ethyl alcohol. The solutionalso can be a frozen form of a liquid solution.

The term “miscible” as used herein refers to the ability of one or morecomponents, such as liquids, solids and gases, to mix together to form asingle, homogeneous phase. Thus, two liquids are miscible if they can bemixed to form a single, homogenous liquid whose distinct components arerecognized only at the molecular level. When components are “partiallymiscible,” it means that they can be mixed to form a single homogenousphase in a certain concentration range, but not at other concentrationranges. As used herein, when a solvent is “partially miscible” withanother solvent, it means that it is miscible at a concentration ofabout or at 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, 1%, 0.5% or below volume/volume (v/v), when mixed withthe other solvent.

As used herein, “immiscible” means that when two or more components,such as liquids, solids or gases are mixed, they form more than onephase. For example, when an organic solvent is immiscible with anaqueous solvent (e.g., hexane and water), the organic solvent is visibleas a distinct layer that does not mix with the layer of aqueous solvent.

As used herein, the term “polypeptide,” means at least two amino acids,or amino acid derivatives, including mass modified amino acids and aminoacid analogs, that are linked by a peptide bond, which can be a modifiedpeptide bond. The terms “polypeptide,” “peptide” and “protein” are usedessentially synonymously herein, although the skilled artisan willrecognize that peptides generally contain fewer than about fifty toabout one hundred amino acid residues, and that proteins often areobtained from a natural source and can contain, for example,post-translational modifications.

A polypeptide or protein can be translated from a polynucleotide, whichcan include at least a portion of a coding sequence, or a portion of anucleotide sequence that is not naturally translated due, for example,to it being located in a reading frame other than a coding frame, or itbeing an intron sequence, a 3′ or 5′ untranslated sequence, a regulatorysequence such as a promoter, or the like. A polypeptide also can bechemically synthesized and can be modified by chemical or enzymaticmethods following translation or chemical synthesis. A polypeptide canbe post-translationally modified by phosphorylation (phosphoproteins),glycosylation (glycoproteins, proteoglycans), and the like, which can beperformed in a cell or in a reaction in vitro.

As used herein, the term “fusion protein” refers to a protein that is aconjugate of domains obtained from more than one protein or polypeptide.A domain can be a polypeptide tag, such as a His6 tag. The conjugatescan be prepared by linking the domains by chemical conjugation,recombinant DNA technology, or combinations of recombinant expressionand chemical conjugation.

A variety of chemical linkers are known to those of skill in the art andinclude, but are not limited to, amino acid and peptide linkages,typically containing between one and about 60 amino acids, moregenerally between about 10 and 30 amino acids, heterobifunctionalcleavable cross-linkers, including but are not limited to,N-succinimidyl(4-iodoacetyl)-aminobenzoate,sulfosuccinimidyl(4-iodoacetyl)-aminobenzoate,4-succinimidyl-oxycarbonyl-a-(2-pyridyldithio)toluene,sulfosuccinimidyl-6-[a-methyl-a-(pyridyldithiol)-toluamido]hexanoate,N-succinimidyl-3-(-2-pyridyldithio)-propionate, succinimidyl6[3(-(-2-pyridyldithio)-propionamido]hexanoate, sulfosuccinimidyl6[3(-(-2-pyridyldithio)-propionamido]hexanoate,3-(2-pyridyldithio)-propionyl hydrazide, Ellman's reagent,dichlorotriazinic acid, and S-(2-thiopyridyl)-L-cysteine.

The term “sialidase fusion protein” as used herein refers to a fusionprotein in which one or more domains is a sialidase or a portion thereofthat retains at least about 60% or 60%, about 70% or 70%, or about or at75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of its catalyticactivity. A sialidase fusion protein as used herein also can refer to afusion protein that contains a protein or polypeptide that issubstantially homolgous to a sialidase and possesses the enzymaticactivity of a sialidase.

The term “catalytic domain” of a protein as used herein refers to aprotein or polypeptide in which the only portion of the sequence that issubstantially homologous to a sialidase is a sequence of amino acidresidues that includes the domain responsible for the catalytic activityof the protein (e.g., residues 274-666 of SEQ ID NO: 1 are identified asthe catalytic domain of Actinomyces viscosus sialidase) or catalyticallyactive fragments thereof. The catalytic domain or catalytically activefragment thereof retains at least about 60% or 60%, about 70% or 70%, orabout or at 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of thecatalytic activity of the protein.

As used herein, the term “flowability characteristic” refers to aproperty that renders the ability to “flow,” where “flow” is a propertythat can permit a substance to be poured and to assume the shape of acontainer that it is poured into, without hindrance due to, for example,aggregation. Fluids generally have the property of “flow,” whichgenerally renders them deformable, i.e., they can change their shape.The term “fluid” as used herein encompasses colloids containing liquids,including emulsions, aerosols and gases. Liquids, aerosols and gaseswith suspensions of solid particles, such as microparticles, also areconsidered “fluid” as defined herein.

As used herein, an emulsion is defined as a colloid of two immiscibleliquids, a first liquid and a second liquid, where the first liquid isdispersed in the second liquid.

As used herein, surfactants (or “surface-active agents”) are chemical ornaturally occurring entities which, when dissolved in an aqueoussolution, reduce the surface tension of the solution or the interfacialtension between two or more phases in solution. The surfactant moleculesgenerally are amphiphilic and contain hydrophilic head groups andhydrophobic tails. The surfactant molecules can act as stabilizersand/or improve flowability characteristics of the microparticlesprovided herein.

As used herein, a combination refers to any association between two oramong more items for a purpose. For example, a combination ofmicroparticles and an inhaler can be used for pulmonary delivery of atherapeutic agent.

As used herein, a composition refers to any mixture. It can be asolution, a suspension, liquid, powder, a paste, aqueous, non-aqueous orany combination thereof.

As used herein, a kit refers to a combination in which components arepackaged optionally with instructions for use and/or reagents andapparatus for use with the combination.

As used herein, the term “enzyme” means a protein that catalyzes achemical reaction or biological process. Enzymes generally facilitateand/or speed up such reactions and processes. In addition, enzymesgenerally are specific for a particular reaction or process, convertinga specific set of reactants into specific products.

As used herein, the term “colloid” refers to a dispersion of solidparticles, such as microparticles, in a liquid, such as the solution inwhich the microparticles are formed. The term “colloidal stability”refers to a colloid in which the particles are not substantiallyaggregated. For example, a stable colloid is one in which about 30%,25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or less of the solidparticles, such as microparticles, have formed aggregates.

The term “agglomerates” refers to the association of one or moreparticles, such as microspheres, loosely held together by van der Waalsforces or surface tension or electrostatic or combinations thereof. Insome instances, associations held by electrostatic forces can be definedas “Flocculates.” For the purposes herein, “Agglomerates” also encompass“Flocculates”. Agglomerates can generally readily be broken apart byshear forces within the air or liquid. The term “disperse” or“dispersivity” refers to the ability of the particles to “flow,” i.e.,the extent to which the movement is not impeded by the presence of, forexample, aggregates.

The term “aggregates” refers to the association of one or moreparticles, such as microspheres, amorhous precipitates, crystal- orglass-like particles or combinations thereof. Aggregates generally arenot easily broken apart which inhibits their ability to disperse or formhomogeneous suspensions or to form aerosols with desirable properties.

The term “non-denatured” as used herein is in reference to proteins andmeans a conformation of a protein, i.e., its secondary structure,tertiary structure, quaternary structure or combinations thereof, whichessentially is unaltered from the protein in its naturally occurringstate. The terms “non-denatured” and “native” are used interchangeablyherein and mean a protein that retains all or at least about 50%, 60%,70%, 80%, 85%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of itslength and/or natural conformation. The terms “non-denatured” or“native” as used interchangeably herein include the natural state of aprotein in a cell, such as it's length and conformation includingsecondary, tertiary and quaternary structures. As defined herein, the“non-denatured” or “native” proteins including those in the compositionsprovided herein generally retain all or at least about 50%, 60%, 70%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of thenormal activity or function of the proteins in their natural state,e.g., as a nutrient to provide amino acid building blocks, anantioxidant, an enzyme, an antibody, a regulator of gene expression, ascaffold, etc.

As used herein, the terms “activity” or “function” are interchangeablewith “biological activity” and refer to the in vivo activities of acompound, such as a protein, vitamin, mineral or drug, or physiologicalresponses that result upon in vivo administration of a compound,composition or other mixture. Activity, thus, encompasses therapeuticeffects and pharmaceutical activity of compounds, compositions andmixtures. Biological activities also can be observed in in vitro systemsdesigned to test or use such activities.

As used herein, “functional activity” also is interchangeable with“activity,” “biological acitivity” or “function” and refers to apolypeptide or portion thereof that displays one or more activitiesassociated with the native or non-denatured protein. Functionalactivities include, but are not limited to, biological activity,catalytic or enzymatic activity, antigenicity (ability to bind to orcompete with a polypeptide for binding to an anti-polypeptide antibody),immunogenicity, ability to form multimers, and the ability tospecifically bind to a receptor or ligand for the polypeptide.

The term “denatured” as used herein refers to a protein that is alteredfrom its native or non-denatured conformation, i.e., its secondary,tertiary or quaternary structure or combinations thereof. The alteredconformation generally occurs by processing steps that includepasteurization, radiation, heat, chemicals, enzyme action, exposure toacids or alkalis, and ion-exchange and any combinations thereof.Denaturation of a protein generally results in diminishing all or some,generally more than 50% and at least about 70%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99%, of the original propertiesincluding activity and function of the protein in its native ornon-denatured state.

As used herein, the term “nutritional supplement” means a substance orcomposition that provides nutrients, including vitamins, minerals, fattyacids, amino acids, carbohydrates, enzymes, proteins, biochemicals andtheir metabolites, herbs and plants, to a host, such as an animal,including a human being. Nutrients that are supplied to the host throughnutritional supplements can include nutrients essential for survival,good health, curing disease or preventing disease that are missing ordeficient in a host's diet, and nutrients that are believed to augmentgood health, prevent disease or cure disease but are not consideredessential for survival or good health.

As used herein, “hydrophobic” refers to a substance that is not chargedor charge-polarized, or is not sufficiently charged or charge-polarizedto bond with water or other polar solvents. Hydrophobic ligands canassociate with each other or with other non-polar molecules or solventsin the presence of water or a polar solvent, through hydrophobicinteractions. A hydrophobic ligand generally also is more soluble innon-polar solvents than in polar solvents. Examples of non-polarsolvents include alkanes such as hexane, alkyl ethers such as diethylether, aromatic hydrocarbons such as benzene and alkyl halides such asmethylene chloride and carbon tetrachloride, mono-, di- andtriglycerides, fatty acids, such as oleic, linoleic, palmitic, stearic,conjugated forms thereof and their esters.

As used herein, the term “therapeutic agent” means an agent which, uponadministration to a host, including humans, effectively ameliorates oreliminates symptoms or manifestations of an inherited or acquireddisease or that cures said disease.

As used herein, “shelf life” or “stability” refers to the time afterpreparation of the microparticle composition that the compositionretains at least about or 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% of the initial protein activity that is present inthe composition and other general physical characteristics ofmicrhospheres such as size, shape, and aerodynamic particle sizedistribution. Thus, for example, a composition that is stable for or hasa shelf life of 30 days at room temperature, defined herein as range ofbetween about 18° C. to about 25° C., 26° C., 27° C. or 28° C., wouldhave at least about 70%, 80%, 85%, 90% 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% of the initial amount of the activity of protein presentin the composition at 30 days following storage at 18° C. to about 25°C., 26° C., 27° C. or 28° C. The shelf life of the microparticlecompositions provided herein generally is at least about 10 days at 55°C., at least about 2-3 weeks at 42° C., and at least about eight monthsor greater at 25° C., however, microparticles compositions of any lengthof shelf life at any temperature that are produced by the methodsprovided herein are contemplated herein.

As used herein, “a biologically active agent, “an active agent,” “abiological agent,” or “an agent,” is any substance which when introducedinto the body causes a desired biological response, such as alteringbody function at the cellular, tissue or organ level and/or alteringcosmetic appearance, such as body weight and shape. Such substance canbe any synthetic or natural element or compound, protein, cell, ortissue including a pharmaceutical, drug, therapeutic, nutritionalsupplement, herb, hormone, or the like, or any combinations thereof. Theterms also encompass pharmaceutically acceptable, pharmacologicallyactive derivatives of those active agents specifically mentioned herein,including, but not limited to, salts, esters, amides, prodrugs, activemetabolites, isomers, fragments, analogs, and the like. When the terms“biologically active agent,” “biological agent” and “agent” are used,then, or when a particular active agent is specifically identified, itis intended to include the active agent per se as well aspharmaceutically acceptable, pharmacologically active salts, esters,amides, prodrugs, active metabolites, isomers, fragments and analogs.

As used herein, a “subject” is defined as an animal, including a mammal,typically a human.

As used herein, “therapeutically effective amount” refers to an amountof the active agent for a desired therapeutic, prophylactic, or otherbiological effect or response when a composition is administered to asubject in a single dosage form. The particular amount of active agentin a dosage will vary widely according to conditions such as the natureof the active agent, the nature of the condition being treated, the ageand size of the subject.

As used herein, “pharmaceutically acceptable derivatives” of a compoundinclude salts, esters, enol ethers, enol esters, acids, bases, solvates,hydrates or prodrugs thereof. Such derivatives can be readily preparedby those of skill in this art using known methods for suchderivatization. The compounds produced can be administered to animals orhumans without substantial toxic effects and either are pharmaceuticallyactive or are prodrugs. Pharmaceutically acceptable salts include, butare not limited to, amine salts, such as but not limited toN,N′-dibenzylethylenediamine, chloroprocaine, choline, ammonia,diethanolamine and other hydroxyalkylamines, ethylenediamine,N-methylglucamine, procaine, N-benzylphenethylamine,1-para-chlorobenzyl-2-pyrrolidin-1′-ylmethylbenzimidazole, diethylamineand other alkylamines, piperazine and tris(hydroxymethyl)aminomethane;alkali metal salts, such as but not limited to lithium, potassium andsodium; alkali earth metal salts, such as but not limited to barium,calcium and magnesium; transition metal salts, such as but not limitedto zinc; and other metal salts, such as but not limited to sodiumhydrogen phosphate and disodium phosphate; and also including, but notlimited to, salts of mineral acids, such as but not limited tohydrochlorides and sulfates; and salts of organic acids, such as but notlimited to acetates, lactates, malates, tartrates, citrates, ascorbates,succinates, butyrates, valerates and fumarates. Pharmaceuticallyacceptable esters include, but are not limited to, alkyl, alkenyl,alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl andheterocyclyl esters of acidic groups, including, but not limited to,carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids,sulfinic acids and boronic acids.

As used herein, “treatment” means any manner in which one or more of thesymptoms of a condition, disorder or disease are ameliorated orotherwise beneficially altered. Treatment also encompasses anypharmaceutical use of the compositions herein, such as use for treatinginfluenza.

As used herein, “organic solvent” refers to a solvent that is an organiccompound, which is any member of a large class of chemical compoundswhose molecules contain carbon and hydrogen. Such solvents can include,for example, compounds from the following classes: aliphatic or aromaticalcohols, polyols, aldehydes, alkanes, alkenes, alkynes, amides, amines,aromatics, azo compounds, carboxylic acids, esters, dioxanes, ethers,haloalkanes, imines, imides, ketones, nitriles, phenols and thiols.

As used herein, an “aqueous solvent” refers to water, or a mixture ofsolvents that contains at least about 50% or 50%, at least about 60% or60%, at least about 70% or 70%, or about or at 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or higher amounts of water. The term “aqueoussolvent” as used herein also refers to solutions containing water as asolvent, such as buffers, salt solutions, solutions containingcounterions, and other solutes that are soluble in water.

As used herein, the term “pI” or “isoelectric point” refers to the pH atwhich there is no net charge on a protein or polypeptide.

As used herein, the term “counterion” refers to a charged orcharge-polarizable molecule that can initiate formation of amicroparticle from a macromolecule, such as a protein, nucleic acid,lipid or oligosaccharide. For example, in the case of the DAS181 fusionprotein (SEQ ID NO:17), sodium sulfate is a counterion because it caninitiate the formation of microparticles in the methods provided herein,whereas glycine, sodium chloride or sodium acetate generally are notsuitable as counterions for DAS181. Whether a charged molecule is acounterion can be determined empirically based on parameters including,but not limited to, the type of protein, the pH, the ionic strength, thetype of organic solvent used, and the presence of salts and additionalingredients such as active agents. As provided and described herein,counterions can be anionic or having a net negative charge orcharge-polarizable group(s), cationic or having a net positive charge orcharge-polarizable group(s), or zwitterionic and possessing bothnegative and positive charged or charge-polairizable groups.

As used herein, the term “cooling” refers to a lowering of temperatureto a desired temperature for obtaining microparticles or, once themicroparticles are obtained, further lowering the temperature to adesired temperature for obtaining dry preparations of the microparticlesby volatilizing solvents (e.g., for freeze-drying). The term “gradualcooling” or “gradually cooling” or “gradually cooled” as used hereinmeans that the lowering of temperature to a desired temperature fromambient temperature (about or at 18° C. to about or at 30° C.) formicroparticle formation occurs at a rate or for an amount of time thatis suitable for generating microparticles in a solution before thesolution becomes frozen. Thus gradual cooling is different from, forexample, snap freezing, spray drying or spray freeze-drying, whereby theentire solution is converted to a solid form without the generation ofdistinct microparticles.

The rate of gradual cooling is empirically determined based on the typeof macromolecule, solvents, counterions and other ingredients as well asthe method of cooling (e.g., a heat exchanger, refrigerator or freezeror freeze-dryer) and can vary, for example, for an amount of time formicroparticle formation of between about or at 1 min, 2 min, 3 min, 5min, 7 min, 10 min, 15 min, 20 min, 25 min, 30 min, 1 h, 2 h, 5 h or 10h to about or at 1.5 min, 2 min, 3 min, 5 min, 7 min, 10 min, 15 min, 20min, 25 min, 30 min, 1 h, 2 h, 5 h, 10 h or 15 h.

As used herein, the term “cooling” refers to a lowering of temperatureto a desired temperature for obtaining microparticles or, once themicroparticles of desired dimensions are obtained, further lowering thetemperature to a desired temperature for obtaining dry preparations ofthe microparticles by volatilizing solvents (e.g., for freeze-drying).The term “gradual cooling” or “gradually cooling” or “gradually cooled”as used herein means that the lowering of temperature to a desiredtemperature from ambient temperature at which the solution cocktail wasformed (about or at −15° C. to about or at 50° C., generally about or at18° C. to about or at 30° C.) for microparticle formation occurs at arate or for an amount of time that is suitable for generatingmicroparticles of desired dimensions in a solution before the solutionbecomes frozen. Thus gradual cooling is different from, for example,snap freezing, spray drying or spray freeze-drying, whereby the entiresolution is converted to a solid form without the generation of distinctmicroparticles.

The rate of gradual cooling is empirically determined based on the typeof macromolecule, solvents, counterions and other ingredients as well asthe method of cooling (e.g., a heat exchanger, refrigerator or freezeror freeze-dryer) and can vary, for example, for an amount of time formicroparticle formation of between about or at 0.1 sec, 0.2 sec, 0.5sec, 1 sec, 10 sec, 20 sec, 30 sec, 40 sec, 50 sec, 1 min, 2 min, 3 min,5 min, 7 min, 10 min, 15 min, 20 min, 25 min, 30 min, 1 h, 2 h, 5 h or10 h to about or at 1.5 min, 2 min, 3 min, 5 min, 7 min, 10 min, 15 min,20 min, 25 min, 30 min, 1 h, 2 h, 5 h, 10 h or 15 h.

Microparticles of desired size also can be formed, for example, byrapidly chilling the cocktail (e.g. using a heat exchanger) and allowingthe suspension of microparticles to be maintained for a certain periodof time without significant temperature changes, then snap freezing thecocktail.

The temperature at which microparticles are formed also is empiricallydetermined based on the type of macromolecule, solvents, counterions andother ingredients as well as the method and uniformity of cooling andcan vary from about or at 15° C., 10° C., 8° C., 5° C., 3° C., 2° C., 1°C., −2° C., −5° C., −7.5° C., −10° C., −15° C., −20° C., −25° C., −30°C., −35° C., −40° C. or −45° C.

As used herein, the term “spray drying” refers to a process wherein asolution containing a macromolecule, such as a protein, is transformedinto a dry particulate form by atomizing into a hot drying medium,generally for a period of about a few milliseconds to 1-2 seconds to afew tens of seconds. The term “spray freeze-drying” as used hereinrefers to a process wherein a solution containing a macromolecule, suchas a protein, is atomized into a cryogenic medium, such as liquidnitrogen, to obtain frozen droplets of solution that can then be driedby lyophilization. The term “snap freezing” or “rapid freezing” or“quick freezing” as used interchangeably herein refers to freezing asolvent or solution, including solutions containing macromolecules, suchas proteins, by immersing the container with good heat transferproperties (e.g. thin-wall glass or metal test tube) holding the solventor solution in liquid nitrogen or pouring the solution directly intoliquid nitrogen. “Snap freezing” and “rapid freezing” generally occurwithin a period of about a few milliseconds to 1-2 seconds to a few tensof seconds.

The term “lyophilize” or “lyophilization” as used herein is synonymouswith “freeze drying” and refers to a process wherein a solution,including an emulsion, colloid or suspension, is frozen and the solventsare volatilized directly into the vapor state, leaving behind the solidcomponents.

B. Methods for Preparing Macromolecular Microparticle Compositions

Provided herein are methods of making microspheres having a high contentof a macromolecule, such as a protein. The microspheres provided hereinare prepared by controlled precipitation in the presence of a counterionand an organic solvent. The microspheres are suitable for preparingpharmaceutical compositions which can be delivered to a patient bydelivery routes including pulmonary, parenteral and oral administrationroutes. The method also can be performed in a batch or continuous mode,for increased efficiency and production.

The microspheres obtained by the methods provided herein are useful asprophylactic, therapeutic or diagnostic agents for treating ordiagnosing disease states in a subject in vivo or in vitro. The sizes ofthe microspheres obtained by the methods provided herein can becontrolled by adjusting parameters including type and concentration oforganic solvent, protein or macromolecule concentration, ionic strength,counterion type and concentration, rate and time of cooling, to providemicrospheres in a wide range of sizes, from 0.001 micron to 50 micronsor greater, that can deliver therapeutic agents via a desired routeincluding pulmonary (e.g., 1 micron to 5 micron particles for deliveryto the throat, trachea and bronchi for treatment of influenza and otherrespiratory infections), subcutaneous, intramuscular, intravenous andother routes (e.g., using particles of tens of microns in size). renderactive components of inhalant medicines for human subjects.

The steps of the method provided herein include: combining the asolution containing the macromolecule with a counterion and an organicsolvent, and gradually cooling the resulting solution to a temperaturewhereby microparticles are formed. In one embodiment, the steps can bedescribed as follows:

1) To a solution containing a macromolecule, such as a protein, nucleicacid, oligosaccharide or lipid, adding a counterion and an organicsolvent at concentrations that do not cause precipitation of themacromolecule at ambient temperature;

2) Precipitation: cooling the macromolecule/solvent cocktail to initiateformation of microspheres; and

3) Dehydration: freezing of the suspension and removal of organicsolvent and water by sublimation (freeze-drying, e.g., at a temperatureof about −20° C. to about −80° C., or about −30° C. to about −80° C., orabout −40° C. to about −80° C., or about −45° C. to about −80° C., orabout −45° C. to about −75° C.).

The above steps of the method can be performed sequentially,intermittently or simultaneously in any order. In one embodiment, thecounterion and the organic solvent are added simultaneously orsequentially in any order to the solution containing the macromolecule,followed by chilling. In other embodiments, the solution containing themacromolecule can be pre-chilled to a temperature suitable formicrosphere formation, prior to adding the counterion and organicsolvent. For example, a prechilled aqueous solution of a macromolecule,such as a protein, can be combined with ammonium sulfate andacetonitrile to form microspheres.

The resulting suspension of microparticles can be converted into a drypowder by further cooling to a temperature below freezing point andsubsequent removal of volatiles (water, organic solvent and wherepossible the counterion) by, for example, sublimation using a standardfreeze dryer.

In one embodiment, the microspheres formed by contacting themacromolecule with a counterion and organic solvent and exposed to lowtemperature, are separated from the suspension by methods includingsedimentation or filtration techniques. After separation from theoriginal precipitation mix, the microspheres can be washed and/orcombined with other materials that improve and/or modify characteristicsof macromolecules and microspheres.

In another embodiment, the microspheres prepared by the methods providedherein do not have a direct therapeutic effect, but serve asmicro-carriers for other therapeutic agent(s) or active agent(s)including nutritional supplements. Therapeutic agents can be added atthe time of precipitation or can be added to the suspension of formedmicrospheres prior to lyophilization. Alternatively, therapeutic agentscan be blended into dry powder consisting of microspheres.

Without being bound by any theory, in one aspect, the methods providedherein can permit the formation of microspheres by: (1) neutralizationof charges on the surface of macromolecule by the counterion and (2)decreased solubility of the macromolecule caused by the combined effectsof added organic solvent and gradual cooling.

By choosing a suitable pH in the range of about or at pH 2.0 to about orat 10.5 or greater, depending on the macromolecule, counterion, andorganic solvent, in the presence of a suitable amount of the counterion,a substantial number of the charged groups, in some embodiment allcharged groups, on the surface of the macromolecule can becomeneutralized. A decrease in the polarity of the solution by adding asuitable organic solvent can then initiate the formation of microspheresby precipitation, phase separation, colloid formation, or other suchmethod.

Alternatively, without being bound by any theory, in some embodiments,the observed phenomenon of the precipitation of microspheres also can beexplained by the kosmotropic (structure forming) effect of counterionsand organic solvents due to interactions with the water molecules of theaqueous solution containing the macromolecule at low temperatures.Regardless of the underlying mechanism, in the methods provided herein,the addition of relatively small amounts of organic solvent andcounterion to an aqueous or other hydrophilic solution containing amacromolecule and cooling of the solution results in the production ofcompositions containing microspheres of the macromolecule(s).

In one embodiment, gradual cooling chilling of the cocktail solution canbe performed by passing the cocktail solution through a heat exchanger.The temperature of the heat exchanger and the flow rate of the cocktailthrough the heat exchanger can be adjusted so that the cocktail iseither pre-chilled prior to formation of the microspheres, or is chilledto a temperature whereby microspheres are formed.

In another embodiment, the microspheres formed by the methods providedherein are concentrated or separated from the suspension by methods suchas sedimentation or filtration techniques. Upon formation of themicrospheres, their growth (size) can be controlled by adjusting theionic strength, polarity, pH, or other parameters of the suspension. Theseparation of microspheres from the liquid phase of the cocktailsolution can be performed by centrifugation, filtration (hollow fiber,tangential flow, etc.), or other techniques. The resulting microspheresor concentrated suspensions thereof can be lyophilized or air dried.

In some embodiments, the microspheres separated from the originalprecipitation mix or the dried microspheres can be reconstituted priorto administration as a therapeutic agent or a carrier, or can besuspended in solutions that contain agents that modify characteristicsof the microspheres. The modifying agents can include but are notlimited to bulking agents, excipients, inactive ingredients, stabilityenhancers, taste and/or odor modifiers or masking agents, vitamins,therapeutic agents, anti-oxidants, immuno-modulators, trans-membranetransport modifiers, anti-caking agents, enteric coating agents, agentsthat confer acid resistance, such as against the acids of the digestivesystem, agents that confer protease resistance, chitosans, polymers, andflowability enhancers.

The formation and characteristics of the microspheres produced by themethods provided herein can empirically be determined by varyingparameters, including: nature and concentration of the macromolecule, pHof the cocktail solution, nature and concentration of the counterion,nature and concentration of the organic solvent, ionic strength and thecooling rate by which gradual cooling is effected. The steps of themethods provided herein render the method amenable to high-throughputscreening, such as in a microplate format, for determining suitablecombinations of macromolecule, organic solvent, counterion, pH, ionicstrength and cooling ramp for the generation of microspheres.

Macromolecules

Macromolecules that can be used to form microspheres according to themethods provided herein include a variety of therapeutic agents,diagnostic agents, nutritional agents and other active agents.Therapeutic agents include antibiotics, vaccines, hematopoietics,anti-infective agents, antiulcer agents, antiallergic agents,antipyretics, analgesics, anti-inflammatory agents, antidementia agents,antiviral agents, antitumoral agents, antidepressants, psychotropicagents, cardiotonics, antiarrythmic agents, vasodilators,antihypertensive agents, antidiabetic agents, anticoagulants, andcholesterol lowering agents. Other examples of suitable macromoleculesinclude proteins, peptides, nucleic acids, carbohydrates, proteinconjugates, viruses, virus particles, and mixtures thereof.

The macromolecules can be characterized by their ability to interactwith the counterion and organic solvent, such as citrate (counterion)and isopropanol (solvent), to form intact, discrete microspherescontaining a high content of macromolecule. The content of themacromolecule in the microspheres can vary from about or at 30%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or greater weight/weight (w/w) of themicrospheres. In some embodiments, the macromolecule content ofmicrosphere is substantially the same as the amount of macromoleculeinitially in solution, prior to forming the microspheres.

The macromolecules used to prepare microspheres by the methods providedherein can include peptides, including polypeptides and proteins,carbohydrates, including polysaccharides and nucleic acids (DNA, RNA orPNA). In some embodiments, the macromolecules are proteins, includingtherapeutic proteins such as DAS181 (the sialidase fusion protein havingthe sequence of amino acid residues set forth in SEQ ID NO:17),alpha1-antitrypsin, PI8, eglin c, Ecotin, aprotinin, recombinant humanDNase, insulin, interferons, recombinant human DNAse (rhDNAse, useful,for example, in the treatment of cystic fibrosis as an inhalationtherapeutic (Genentech); see also Shak et al., Proc. Natl. Acad. Sci.USA, 87:9188-9192 (1990)), human serum albumin, human growth hormone,parathyroid hormone and calcitonin. In some embodiments, the protein isDAS181, the counterion is sodium sulfate or sodium citrate, and theorganic solvent is isopropanol.

The methods provided herein can avoid the use of conditions, such asheat, that can denature the protein and reduce its activity. Themicrospheres provided according to the methods provided herein thereforecan be used to prepare vaccines or other therapeutic medications thatrequire proteins or peptides to be present in their native conformation.

The concentration of the macromolecule in solution, used duringprecipitation of the microspheres, can be between about or at 0.1 mg/mlto about or at 0.2, 05, 0.8, 1.0, 2.0, 5.0, 10.0, 12.0, 15.0, 20.0,25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 60.0, 70.0, 80.0, 90.0, 100, or 200mg/ml. In some embodiments, the concentration is between about or at 1mg/ml and about or at 20 mg/ml. Depending on the characteristics of themacromolecule (pI, hydrophobicity, solubility, stability, etc.) andother process parameters, the concentration of macromolecule canempirically be determined to achieve formation of microspheres of adesired size. In general, macromolecules with lower solubility in thesolvent (generally, aqueous solvent) prior to adding counterion andorganic solvent can be used at lower concentrations (0.1-5 mg/ml) toform microspheres according to the methods herein, while macromoleculeswith higher solubility can be used at 1-20 mg/ml or higher. If theformation of amorphous aggregates or aggregated microspheres isobserved, the concentration of the macromolecule generally should bedecreased to reduce or prevent such aggregation.

Nature and Concentration of Counterion

The counterion can be any compound capable of neutralizing one or moreoppositely charged groups on the macromolecule at the pH at which themethod is performed. Depending on the characteristics of themacromolecule (pK, pI, nature and quantity of charged groups,distribution of charge groups on the surface, solubility and structuralstability under different pH conditions), the pH can empirically bedetermined for microsphere formation. In general, if precipitation isperformed at a pH below the pK of the macromolecule, anionic counterionscan be used. In general, if precipitation is performed at a pH above thepK of the macromolecule, cationic counterions can be used. Thecounterion can empirically be selected based on its suitability toinitiate microsphere formation. In some embodiments, the counterion canhave a molecular weight of 60 Da or greater, or about 75 Da or greater.

The counterions can be anionic, cationic or zwitterionic. Anioniccounterions can be inorganic (phosphate, sulphate, thiocyanate,thiosulfate, hypochlorate, nitrate, bromine, iodine, etc.) or organiccompounds that carry charge-polarizable groups including enol, hydroxy,—SH, carboxylic, carboxymethyl, sulfopropyl, sulfonic, and phosphoric.Organic compounds carrying other anionic groups or having negativecharge due to other molecular characteristics also can be used.Compounds that can be used as anionic counterions also include, but arenot limited to, the following: oxaloacetate, malate, maleate, oxalate,piruvate, citrate, succinate, fumarate, ketoglutarate,butanetricarboxylic acid, hydromuconic acid, cyclobutanedicarboxylicacid, dimethyl maleate, deoxyribonucleic acid, polyglutamic acid, folicacid, lactic acid, ascorbic acid, carminic acid, sorbic acid, malonicacid, EDTA, MOPS, TES, MES, PIPES, pyridine, tricine, glycine,glycylglycine, betaine, sulfuric acid, thiosulfuric acid, phosphoricacid, adenosine triphosphate, nitric acid, itaconic acid, pivalic acid,dimethylmalonic acid, and perchloric acid. In some embodiments,itaconic, pivalic, dimethylmalonic, and succinic acids are used ascounterions in the methods provided herein.

Cationic counterions can be inorganic (ammonium, phosphonium, sulfonium,cesium, rubidium, etc.) or organic compounds that carry groups known asamine, amide, imine, imide, guanidine, imidazole, dioxane, aniline.Organic compounds carrying other cationic groups or have positive chargepolarizability due to other molecular characteristics also can be used.Compounds that can be used as cationic counterions also include, but arenot limited to, the following: Tris, Bis-Tris, Bis-Tris propane,diaminopropane, piperazine, piperadine, pentylamine, diaminobutane,propylamine, trimethylamine, triethylamine, spermine, spermidine,putrescine, cadaverine, ethanolamine, diethanolamine, triethanolamine,imidazole, tetramethylammonium, trimethylammonium, ammonium, cesium,rubidium, imidazole, polyethileneimine, DEAE, TEAE, QAE.

Zwitterionic counterions possessing any charged groups in anycombination can also be used. Compounds that can be used as zwitterioniccounterions include, but are not limited to, the following: HEPES,BICINE, glycine, glycylglycine, 6-aminohexanoic acid, piperidic acid,natural and non-natural amino acids (e.g., histidine, glutamine,arginine, lysine).

The counterions can be used as acids (e.g. sulfuric acid) or bases (e.g.imidazole) or their salts (e.g. sodium sulfate or imidazole-HCl).Counterions that can be used in the methods provided herein includethose listed by the National Formulary, United States Pharmacopeia,Japanese Pharmacopeia, or European Pharmacopeia, the clinical safety ofwhich has been demonstrated (citric acid, malic acid, amino acids,sulfate, etc.). In some embodiments, counterions used in the methodsprovided herein include ones for which safety has been established or asfalling into the GRAS (generally regarded as safe) category. Thecounterions (or their salts) can be solid at room temperature (about 25°C.), or at the intended temperature of use and storage). Combinations oftwo or more counterions also can be used. Volatile and liquidcounterions also can be used in the methods provided herein.

The concentration of counterion generally is maintained between about0.1 mM and about 0.2, 0.5, 0.8, 1.0, 2.0, 3.0, 5.0, 7.0, 10.0, 15.0,20.0, 30.0, 40.0, 50.0, 60.0, 70.0, 80.0, 90.0 and 100.0 mM. In someembodiments, the concentration of the counterion is between about or at0.5 mM and about or at 20 mM. Depending on the characteristics ofmacromolecule (pI, hydrophobicity, solubility, stability, etc.) andother process parameters, the concentration of the counterion canempirically be determined using, for example, a high-throughput formatas provided herein. In general, the formation of oversized microspheres,amorphous aggregates or aggregated microspheres indicates that theconcentration of counterion should be decreased, while failure to formmicrospheres (broken glass-like crystals or flakes) or formation ofmicrospheres below the desired size indicates that the concentration ofcounterion should be increased.

Nature and Concentration of Organic Solvent

An organic solvent added to the cocktail in the methods provided hereingenerally can be water miscible and selected from among alcohols(methanol, ethanol, 1-propanol, isopropanol, butanol, tert-bulylalcohol), chloroform, dimethyl chloride, polyhydric sugar alcohols(glycerin, erythriol, arabitol, xylitol, sorbitol, mannitol), aromatichydrocarbons, aldehydes, ketones, esters, ethers (di-ethyl ether),alkanes (hexane, cyclohexane, petroleum ether), alkenes, conjugateddienes, toluene, dichloromethane, acetonitrile, ethyl acetate, polyols,polyimids, polyesters, polyaldehydes, and mixtures thereof. In someembodiments, the organic solvent can be volatile. In other embodiments,when incorporation of the organic solvent into the microspheres isdesired, non-volatile organic solvents can be used that provide, forexample, novel characteristics to the microspheres (e.g., sustainedrelease or added mechanical strength). The concentration of the organicsolvent generally can be maintained between about or at 0.1%, to aboutor at 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40% or 50%,volume/volume (v/v). In some embodiments, the concentration of theorganic solvent is between about or at 1% to about or at 30%, v/v.Organic compounds that are partially miscible or completely immisciblewith water also can be used.

Organic solvents that can be used in the methods provided herein includealcohols and others listed as Class 3 and 2 solvents in InternationalConference on Harmonisation (ICH) Harmonised Tripartite Guideline(Impurities: Guideline for Residual Solvents), safe handling of whichhas been established in pharmaceutical and food industries.

Depending on the characteristics of the macromolecule (hydrophobicity,solubility, stability, etc.) and other process parameters, the choiceand concentration of the organic solvent can be optimized, for example,using high-throughput screening on microtiter plates or similar chips orother device. In general, uncontrolled precipitation before theinitiation of cooling, the formation of oversized microspheres,amorphous aggregates, aggregated microspheres or sticky aggregatesindicates that the concentration of organic solvent should be decreased,while failure to form microspheres (broken glass-like crystals orflakes) or formation of microspheres below the desired size indicatesthat the concentration of the organic solvent should be increased.

pH

In addition to initiating microsphere formation, the counterion also canserve as a buffer. Alterenately, in some embodiments, a bufferingcompound can be used to obtain the desired pH. In some embodiments, thebuffering compound is 60 Da or larger. Depending on the characteristicsof the macromolecule (pI, hydrophobicity, solubility and stability at aspecific pH, etc.) and other process parameters, the optimal pH canempirically be adjusted to achieve formation of microspheres of desireddimensions and preserve the activity of the macromolecule. In general,failure to form microspheres (broken glass-like crystals or flakes)indicates that the protein may be too soluble under the conditions used.Formation of amorphous aggregates can indicate that precipitation is notwell controlled and the protein may not be stable or soluble at the pHused.

When the macromolecule is a protein, it has been observed that certainprotein/counterion combinations can cause immediate and uncontrolledprecipitation at certain pH values. The high-throughout screeningmethods provided herein can be used to empirically determine theappropriate combination of protein, pH and counterion to formmicrospheres of desired dimensions. his is easily remedied by changingthe pH of the cocktail, by using a different counterion or by decreasingconcentration of the protein in cocktail. In general, for formingprotein-based microspheres, a pH value that is below the pI of theprotein provides optimal microsphere formation

Ionic Strength

The ionic strength of the cocktail solution can be modulated by theconcentration of the counterion or by other salts such as chlorides oracetates. In some embodiments, no additional salt is required to producemicrospheres. In certain embodiments, the ionic strength can be adjustedto preserve the structural integrity and activity of the macromolecule.Examples of other applications where the presence of specific salts canbe beneficial include formulations of parenteral and other drugs, orfoods where specific tonicity or buffering capacity may be required uponreconstitution of microspheres.

Cooling Ramp

The cocktail containing a macromolecule, a counterion and an organicsolvent initially is prepared, prior to cooling, at a temperature atwhich the macromolecule is soluble, generally about −15° C. to about 30°C. In some embodiments, the initial temperature, prior to cooling is atambient temperature (18-25° C.). The microspheres are formed by aprocess such as precipitation, phase separation or colloid formationupon gradual cooling to a temperature below the temperature at which themacromolecule is dissolved and in solution. The rate at which cooling isperformed can control the formation and other characteristics such assize of the microspheres. In general, when the macromolecule is protein,flash-freezing in liquid nitrogen does not generate microspheres

The rate at which cooling and freezing of the cocktail (cooling ramp) isperformed can determine the final size of the microspheres. In general,a faster cooling ramp yields smaller microspheres whereas a slowercooling ramp yields larger microspheres. Without being bound by anytheory, the cooling rate can determine the rate of: (1) nucleation thatproduces initial smaller microspheres and (2) a fusion process in whichthe initial microspheres coalesce (aggregate) and anneal into largermicrospheres. Fusion of the smaller particles into larger ones is a timedependent process that can be determined, for example, by the durationfor which liquid suspension of microspheres exists prior to freezing.Due to the reversible nature of the bonds between certainmacromolecules, such as some proteins, in the microsphere compositionsprovided herein, smaller microspheres annealing into larger particlescan generate microspheres with smooth surfaces. Depending on the size ofmicroparticles desired, the cooling rate can be from about 0.01° C./minor 0.01° C./min to about 20° C./min or 20° C./min; from about or at0.05° C./min or about or at 0.1° C./min to about or at 10° C./min orabout or at 15° C./min, from about or at 0.2° C./min to about or at 5°C./min, from about or at 0.5° C./min to about or at 2° C./min, or aboutor at 1° C./min. In some embodiments, the cooling ramp can be between0.1° C. per minute and about 40° C. per minute. In other embodiments, acooling ramp can be between about 0.5° C. per minute and 15° C. perminute.

Depending on the specific needs, in some embodiments it can be desirableto adapt the production process to the specific equipment. In someembodiments, a lyophilizer with temperature-controlled shelves can beused for the cooling. if the microspheres produced are larger thandesired, other parameters of the process including concentration ofmacromolecule, organic solvent, counterion, ionic strength and/or pH canbe modified to achieve the desired reduction in size of themicrospheres.

For a faster cooling ramp (smaller particle size), the cocktail solutioncan be passed through a heat exchanger, such as that used in acontinuous mode. If the size of microspheres needs to be increased,increased concentrations of one of the cocktail ingredients(macromolecule, organic solvent, counterion) can provide the desiredincrease in the size of microspheres.

In general, the cooling should be performed uniformly and at a steadyrate to prevent the formation of aggregates and crystal. Depending onthe concentration of the organic solvent, the precipitation of themacromolecule into microspheres can occur in several ways. At higherconcentrations of organic solvent (about 5%-40%, dependent on the actualcomponents used) the microspheres generally can form when the cocktailsolution is still in liquid form. At lower concentrations of organicsolvent (2-25%, dependent on the actual components used) ice crystalscan form first, following which the expelled macromolecules and organicsolvent reach can reach a critical local concentration and precipitate.A further decrease of temperature in the near-bottom layer of thelyophilizer tray can leas to complete solidification of the liquidsuspension and further expulsion of the organic solventinto the toplayer. An excess of organic solvent in the top layer can causeuncontrolled precipitation of the macromolecule and aggregation ofmicrospheres. This effect usually can be alleviated by selectingappropriate ratios of the components—macromolecule, counterion, organicsolvent, salts, etc. in the cocktail. In addition, maintaining a thinlayer of cocktail in the lyophilization tray or mixing of the cocktailwhile being chilled can prevent formation of aggregates and crystals andyield uniform microspheres. For example if a relatively lowconcentration of Isopropanol (e.g. 2-6%) is used, and a thin layer ofcocktail (10-20 mm) is filled into the tray, and the tray is placed on apre-chilled shelf (−30-75° C.) uniform microspheres can be obtained.

The methods provided herein can lead to substantially all or all theprotein or other macromolecule being incorporated from the solution intothe microspheres

High-Throughout Screening of Microparticle Formation Conditions andOptimization of Particle Formation

Depending on the characteristics of the macromolecule, the compositionof the cocktail solution used to prepare the microspheres according tothe methods provided herein can be optimized. The optimization canrapidly be performed in a medium or high throughput format using, forexample microtiter plate(s) or chips where tens to hundreds to thousandsto tens of thousands of cocktails can be screened simultaneously. Insome embodiments, a number of pH values in conjunction with cationic,anionic or zwitterionic counterions and organic solvents at variousconcentrations can be screened. For example, the screening can beperformed using several identical microtiter plates, to each of whichthe macromolecule of interest is added at various concentrations. Eachset of test conditions can be screened in duplicate. In someembodiments, microplates with flat-bottom well can be used with theskirt of the microtiter plate broken off to permit good heat transferbetween the lyophilizer shelf and the bottoms of the wells. Themicroplates can be placed on the shelves of the lyophilizer and cooledto form microspheres and to subsequently solidify the suspensions. Uponfreezing of the contents of the wells, a vacuum can applied. At the endof lyophilization, one of the duplicate plates can be reconstituted withwater or a buffer of choice to observe if certain conditions renderedthe macromolecule insoluble or reduced its activity. Conditions thatresulted in material that can readily be resolubilized or providemicrospheres with desirable characteristics can be subjected to furtheranalysis by spectroscopic, chromatographic, enzymatic or other assays toconfirm that native structure and activity are preserved. Lyophilizedmaterial in a duplicate plate can be used for microscopy to determinewhether microspheres are formed. Conditions that produced microspherescan further be modified and fine-tuned to produce microspheres ofdesirable size and characteristics.

Kits for performing high-throughput screens can be provided and cancontain all the ingredients used in the methods provifef hereinincluding one or more of a macromolecule, such as a protein, buffers,pre-dispensed cocktail of known composition (organic solvent,counterion) and/or salts. Kits can contain 3, 4, 5, 10, 15, 20, 30, 40,50, 100 or more (typically 96 or more) buffers with predetermined pH,counterion, ionic strength and organic solvent in each microtiter plate.The microtiter plate supplied with the kit can be modified so thatbottoms of wells are in direct contact with the shelf of lyophilizer.

C. Macromolecular Microparticle Compositions

The macromolecules contained in the microparticle compositions obtainedby the methods provided herein are substantially structurally andchemically unchanged by the methods. For example, when the macromoleculeis Green Fluorescent Protein or Red Fluorescent Protein, theirfluorescence and native conformation and activity of the proteins areretained in the microparticles. The dry microspheres, obtained byvolatilizing substantially all of the solvents and/or moisture exceptfor the solvent and other components associated with the microspheres,can be stored and their activity can substantially be recovered uponreconstitution. The relatively low moisture content of themicroparticles provided herein, for example, between about or at 0.1% toabout or at 0.2%, 0.3%, 0.5%, 1.0%, 2.0%. 3.0%. 4.0%, 5.0%, 5.5%, 6.0%,6.5%. 7.0%. 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%,12.0%, 12.5%, 14%, 15%, 16%, 17%, 18% 19%, or 20%, can provide improvedstability. The microspheres obtained by the methods provided herein alsoare homogeneous in size and shape, and can be obtained reproducibly withthe desired characteristics. Other techniques traditionally used forpreparation of dry formulations (salt precipitation, alcohol or acetoneprecipitation, lyophilization, e.g.) can result in complete or partialdenaturation of the macromolecules, such as proteins. In addition, themicrospheres prepared by the methods provided herein avoid the need forcomplex or specialized spray drying, spray freeze-drying, supercriticalfluid anti-solvent based processes or milling processes (See, forexample, Laube B L. The expanding role of aerosols in systemic drugdelivery, gene therapy, and vaccination. Respir Care 2005;50(9):1161-1176; Taylor G, Gumbleton M. Aerosols for MacromoleculeDelivery: Design Challenges and Solutions. American Journal of DrugDelivery 2004; 2(3):143-155; Smyth H D C, Hickey A J. Carriers in DrugPowder Delivery. Implications for Inhalation System Design. AmericanJournal of Drug Delivery 2005; 3(2):117-132; Cryan S A. Carrier-basedstrategies for targeting protein and peptide drugs to the lungs. AAPS J2005; 7(1):E20-E41; LiCalsi C, Maniaci M J, Christensen T, Phillips E,Ward G H, Witham C. A powder formulation of measles vaccine for aerosoldelivery. Vaccine 2001; 19(17-19):2629-2636; Maa Y F, Prestrelski S J.Biopharmaceutical powders: particle formation and formulationconsiderations. Curr Pharm Biotechnol 2000; 1(3):283-302; Maa Y F,Nguyen P A, H su SW. Spray-drying of air-liquid interface sensitiverecombinant human growth hormone. J Pharm Sci 1998; 87(2):152-159;Vanbever R, Mintzes J D, Wang J et al. Formulation and physicalcharacterization of large porous particles for inhalation. Pharm Res1999; 16(11):1735-1742; Bot A I, Tarara T E, Smith D J, Bot S R, Woods CM, Weers J G. Novel lipid-based hollow-porous microparticles as aplatform for immunoglobulin delivery to the respiratory tract. Pharm Res2000; 17(3):275-283; Maa Y F, Nguyen P A, Sweeney T, Shire S J, Hsu C C.Protein inhalation powders: spray drying vs spray freeze drying. PharmRes 1999; 16(2):249-254; Sellers S P, Clark G S, Sievers R E, CarpenterJ F. Dry powders of stable protein formulations from aqueous solutionsprepared using supercritical CO(2)-assisted aerosolization. J Pharm Sci2001; 90(6):785-797; Garcia-Contreras L, Morcol T, Bell S J, Hickey A J.Evaluation of novel particles as pulmonary delivery systems for insulinin rats. AAPS PharmSci 2003; 5(2):E9; Pfutzner A, Flacke F, Pohl R etal. Pilot study with technosphere/PTH(1-34)—a new approach for effectivepulmonary delivery of parathyroid hormone (1-34). Horm Metab Res 2003;35(5):319-323; Alcock R, Blair J A, O'Mahony D J, Raoof A, Quirk A V.Modifying the release of leuprolide from spray dried OED microparticles.J Control Release 2002; 82(2-3):429-440; Grenha A, Seijo B,Remunan-Lopez C. Microencapsulated chitosan nanoparticles for lungprotein delivery. Eur J Pharm Sci 2005; 25(4-5):427-437; Edwards D A,Hanes J, Caponetti G et al. Large porous particles for pulmonary drugdelivery. Science 1997; 276(5320):1868-1871; McKenna B J, Birkedal H,Bartl M H, Deming T J, Stucky G D. Micrometer-sized spherical assembliesof polypeptides and small molecules by acid-base chemistry. Angew ChemInt Ed Engl 2004; 43(42):5652-5655; Oh M, Mirkin C A. Chemicallytailorable colloidal particles from infinite coordination polymers.Nature 2005; 438(7068):651-654; U.S. Pat. No. 5,981,719; U.S. Pat. No.5,849,884 and U.S. Pat. No. 6,090,925; U.S. Patent application No.20050234114; U.S. Pat. No. 6,051,256).

The microparticles obtained by the methods provided herein can be of anyshape and can have sizes (mean width or diameters) in the range of fromabout or at 0.001 micron to about or at 0.002, 0.005, 0.01, 0.02, 0.03,0.05, 0.1, 0.02, 0.03, 0.5, 1.0, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5,6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 15.0, 20.0, 25.0, 30.0,35.0, 40.0, 45.0, or 50.0 or greater microns. For pulmonaryadministration to the alveoli, the size can be from about 0.1 micron orless to about 0.5 micron. For pulmonary administration to the throat,trachea and bronchi, the size can be from about or at 0.5 microns toabout or at 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or6.5 microns, or in some embodiments from about or at 1.0 micron to aboutor at 2.0 microns. In some embodiments, the microparticles aresubstantially spherical in shape.

The macromolecules that can be used to form microparticles according tothe methods provided herein can include therapeutic and diagnosticagents, processed foods, dietary supplements and polymers. In someembodiments, cross-linking agents, salts, or other compounds can beincluded in the formulation cocktail to modify solubility of themicrospheres and/or enhance their mechanical strength. In someembodiments, microspheres that are insoluble in most aqueous or organicsolvents can be used to manufacture particles such as chromatographicresins and dispersible abrasives. In other embodiments, microsphereswith partial solubility in solvents such as pharmaceutical vehicles fordelivery can be useful in the manufacture of sustained release activeagent or therapeutic formulations.

In some embodiments, the microparticles provided herein can be used incombination with an inhaler device to deliver a therapeutic dose ofmacromolecular microspheres to the respiratory airways and lungs of asubject. For example, when the macromolecule is the DAS181 protein(sequence set forth in SEQ ID NO: 17), microspheres of about 0.5 micronto about 8 microns, or about 1 micron to about 5 micron can be obtainedby the methods provided herein, using sodium sulfate as the counterionand isopropanol as the organic solvent. For DAS181 microspheres, whichare administered to prevent or treat viral infections that initiate inthe respiratory tract, such as influenza, it can be desirable to depositthe microspheres in the throat, trachea or bronchi. The DAS181 fusionprotein formulated as microspheres can act by degrading the receptorsialic acids in the throat/trachea/bronchi, thus preventing viralbinding and infection at these sites. For optimal delivery of the DAS181microspheres to sites where respiratory viral infection can beinitiated, i.e., in the throat, trachea or bronchi, the microspheresmust not be (a) so big that they are trapped at the front end in themouth (i.e., microspheres are too big, about 8 microns or greater); or(b) so small that they are absorbed deep in the lungs and absorbedsystemically into the blood stream through the alveoli where they arenot active and/or can be toxic (i.e., 0.5 micron or smaller). Fordelivery of the DAS181 microspheres to the throat, trachea and bronchi,a size range of about 1 micron to about 5.5-6 microns generally can besuitable.

The inhaler can be used to treat any medical condition in which theprotein or other macromolecule can be administered by inhalationtherapy. Typical inhaler devices can include dry powder inhalers,metered dose inhalers, and electrostatic delivery devices. Typicalapplications of the delivery apparatus of include the deep lung deliveryof insulin and other therapeutic proteins.

In some embodiments, the microspheres obtained by the methods providedherein also can be delivered by oral ingestion, intranasally,intravenously, intramuscularly, subcutaneously, and by other deliverymethods suitable for the delivery of therapeutic molecules. Themicrosphere formulations for pulmonary delivery generally can be in asize range of about 0.5 micron to about 5-6 microns, while thosedesigned for other types of delivery, such as subcutaneous delivery,parenteral delivery or intramuscular delivery can be in a range of fromabout or at 10 micron to about or at 30, 40 or 50 microns.

In some embodiments, the microspheres provided herein have no directtherapeutic effect but can serve as micro-carriers for other therapeuticagent(s). Examples of macromolecules useful for preparation of suchmicrospheres include but are not limited to polysaccharides, glycans,proteins, peptides, polymers or combinations thereof. Therapeutic agentsor other active agents can be added at the time of microsphere formationor added to the suspension of formed microspheres. Alternatively,therapeutic agents can be blended with the dry microsphere compositionsby mixing, tumbling or other techniques practiced in pharmaceutical andfood industries.

In some embodiments, cross-linking agents, lipophilic substances, saltssuch as those with poor solubility in aqueous solvents, or combinationsthereof or other compounds can be included in the formulation cocktailsolution to modify the solubility of the microspheres and/or enhancetheir mechanical strength. Slow dissolution of the microspheres can beuseful in sustained release of therapeutics delivered by oral ingestion,inhalation, intranasally, intravenously, intramuscularly,subcutaneously, and by other delivery methods suitable for the deliveryof therapeutic molecules. In some embodiments, the microspheres can bedelivered by oral ingestion in a form of a pill or capsule with anenteric coating, endocytosed from the duodenum, and the macromoleculereleased into the blood stream or other site of action.

In some embodiments, the microspheres can be rendered insoluble bypartial denaturation of the macromolecule, which upon delivery becomesrenatured and bioavailable.

In other embodiments, the microspheres are substantially spherical inshape, and can have mean diameters within the range of from about 0.1microns to 30.0 microns. In yet other embodiments, the mean diameter ofthe microspheres can be within the range of from about 0.5 microns to5.0 microns, or from about 1.0 microns to 2.0 microns.

In yet another aspect, provided herein are devices and methods fordelivering the microspheres to a subject, such as an animal or humanpatient in need of medical treatment. Suitable delivery routes caninclude parenteral, such as i.m., i.v. and s.c., and non-parenteral,such as oral, buccal, intrathecal, nasal, pulmonary, transdermal,transmucosal, and the like delivery routes. Delivery devices can includesyringes, both needleless and needle containing, and inhalers.

The delivery devices can contain a single dose of the microspheres fortreating a condition that is treatable by rapid or sustained release ofthe macromolecule in vivo. The number of microspheres present in thesingle dose is dependent on the type and activity of the macromolecule.The single dose can be selected to achieve sustained release over aperiod of time that has been optimized for treating the particularmedical condition. For example, when the macromolecule is the DAS181fusion protein (SEQ ID NO:17), the delivery dosage of microspherecompositions containing DAS181 can be from between about 0.5 mg proteinper dose to about 100 mg protein per dose, or about 0.75 mg, 1 mg, 1.5mg, 2 mg, 3 mg, 5 mg, 10 mg, 15 mg, 20 mg, 30 mg, 40 mg, 45 mg, 50 mg,55 mg or 60 mg protein per dose.

The macromolecule component of the microsphere can be any moleculecapable of forming microspheres according to the methods providedherein. In some embodiments the macromolecule is a protein, includingenzymes and recombinant proteins, peptides, carbohydrates,polysaccharides, carbohydrate- or polysaccharide-protein conjugates,nucleic acids, virus, virus particles, conjugates of small molecules(such as a hapten) and proteins, or mixtures thereof. An organic orinorganic natural or synthetic pharmaceutical compound or drug can beincorporated into the microspheres by attaching the drug to amacromolecule, such as a protein, and then forming the microspheres fromthe macromolecule-drug complex or conjugate. It will be understood bythose skilled in the art that a compound incapable of having a tertiaryand quaternary structure can be formed into a microsphere byincorporation or coupling of the compound into a carrier molecule thathas a tertiary and quaternary structure. It will further be understoodby those of skill in the art that the macromolecule can be a portion ofa molecule such as, for example, a peptide, a single-stranded segment ofa double-stranded nucleic acid molecule, or a virus particle, or othermacromolecule having a tertiary and quaternary structure.

The term “macromolecule” also can include a plurality of macromoleculesand includes combinations of different macromolecules such as acombination of a pharmaceutical compound and an affinity molecule fortargeting the pharmaceutical compound to a tissue, organ or tumorrequiring treatment. An affinity molecule can be, foe example, a ligandor a receptor. Examples of ligands can include viruses, bacteria,polysaccharides, or toxins that can act as antigens to generate animmune response when administered to an animal and cause the productionof antibodies.

In some embodiments, the macromolecule is a therapeutic proteinincluding, but not limited to, a sialidase, a sialidase fusion protein,a fusion protein containing a sialidase catalytic domain fused to aGAG-binding domain, a protease, a protease inhibitor, insulin,interferons, human growth hormone, calcitonin, rhDNase or parathyroidhormone, and the protein content of the microspheres can be from aboutor at 50% to about or at 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or greater. For pulmonary administration, the microspherescan have an average size in the range of from about or at 0.5 microns toabout or at 5.0 microns, and in some embodiments, between about or at 1micron and about or at 2 microns.

Other proteins that can be used to form microspheres by the methodsprovided herein can include, but are not limited to, therapeuticproteins including DAS181 (DAS181; SEQ ID NO:17), al-antitrypsin,Ecotin, eglin c, serpin, Pulmozyme (rhDNase), Betaxolol™, Diclofenac™,doxorubicin, acetyl cysteine, Rifampin™, leuprolide acetate, luteinizinghormone releasing hormone (LHRH), (D-Tryp6)-LHRH, nafarelin acetate,insulin, sodium insulin, zinc insulin, protamine, lysozyme,alpha-lactalbumin, basic fibroblast growth factor (bFGF),beta-lactoglobulin, Trypsin, calcitonin, parathyroid hormone, carbonicanhydrase, ovalbumin, bovine serum albumin (BSA), human serum albumin(HSA), phosphorylase b, alkaline phosphatase, beta-galactosidase, IgG,fibrinogen, poly-L-lysine, IgM, DNA, desmopressin acetate, growthhormone releasing factor (GHRF), somatostatin, antide, Factor VIII,G-CSF/GM-CSF, human growth hormone (hGH), beta interferon, antithrombinIII, alpha interferon, alpha interferon 2b.

An inhaler device can be used to deliver a therapeutic protein such asthose listed above or other macromolecule-based microspheres to therespiratory airways and lungs of a subject. The protein microspheres canbe prepared, for example by contacting an aqueous solution of theprotein with a carboxylic acid such as citrate, or sulfate or othercounterion and an organic solvent such as isopropanol, and cooling thesolution to form the microspheres. The protein can be a therapeuticprotein, such as a sialidase, a protease inhibitor, insulin, humangrowth hormone, calcitonin, rhDNase or parathyroid hormone, and theprotein content of the microspheres can be about or at 70% to about orat 90% or more, 95% or more, or at least about 99% or more. Forpulmonary administration, the microspheres, for example DAS181microspheres, can be sized to have a mean diameter in the range of fromabout 0.5 microns to 5.0 microns, or between about 1 micron to about 2microns.

Incubation conditions for forming the microspheres can be optimized toincorporate at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% or greater of the total amount ofmacromolecule present in the solution prior to formation of themicrospheres, by adjusting parameters includign pH, temperature,concentration of macromolecule, or duration of reaction or incubation.

In some embodiments, a molecule or compound that does not producemicrospheres of desirable characteristics, can be incorporated intomicrospheres having desirable characteristics, e.g., of size, deliveryprofile, mechanical strength, by incorporation or coupling of thecompound with a carrier molecule that can form microspheres withdesirable characteristics. In some embodiments, the carriermacromolecule is a protein, and the molecule or compound is bound insideand/or on the surface of the microsphere. In some embodiments, themolecule or compound also can serve as the counterion and initiateand/or facilitate the formation of microspheres.

When preparing microspheres containing a protein, a protein stabilizersuch as glycerol, fatty acids, sugars such as sucrose, ions such aszinc, sodium chloride, or any other protein stabilizers known to thoseskilled in the art can be added prior to cooling the cocktail duringmicrosphere formation, n to minimize protein denaturation.

In some embodiments the microspheres can further be coated on thesurface with suitable molecules and/or coating agents, such as thosethat lend resistance to acids, such as digestive acids, or proteases. Inother embodiments, the microspheres can be non-covalently coated withcompounds such as fatty acids or lipids. The coating can be applied tothe microspheres by immersion in the solubilized coating substance, thenspraying the microspheres with the substance, or by using other methodsknown to those of skill in the art. In some embodiments, the fatty acidsor lipids are added directly to the microsphere-forming cocktailsolution.

Formation of the microspheres by decreasing temperature can be performedby a multitude of conventional methods in batch or continuous modes.Microsphere formation can further be triggered by other methodsincluding, but not limited to, modulating atmospheric pressure, g-forceor surface expansion, including seeding. Microsphere formation can occurimmediately upon exposure to these conditions or can require an extendedperiod of time as provided herein.

Proteins

Exemplary proteins that can be used to form microparticles by themethods provided herein are described below

Sialidases

Sialidases, also referred to as neuraminidases andN-acylneuraminosylglycohydrolases, are a family exoglycosidases thatcatalyze the removal of terminal sialic acid residues fromsialo-glycoconjugates. Sialic acids are a family of α keto acids with9-carbon backbones that are usually found at the outermost positions ofthe oligosaccharide chains attached to glycoproteins and glycolipids.These molecules are involved in a variety of biological functions andprocesses, such as the regulation of innate immunity, cell adhesion, andthe interaction between inflammatory cells and target cells, possiblymediated through the binding of various lectins (Varki et al. (1992)Curr Opin Cell Biol 4:257-266). Sialic acids also are excellent sourcesof carbon, nitrogen, energy, and precursors of cell wall biosynthesis.Further still, sialic acids on eukaryotic cells can be used as receptorsor coreceptors for pathogenic microorganisms, including, but not limitedto, influenza virus, parainfluenza virus, some coronavirus and rotavirusHaemophilus influenzae, Streptococcus pneumonia, Mycoplasma pneumoniae,Moaxella catarrhalis, Helicobacter pylori and Pseudomonas aeruginosa.The most prominent member of the sialic acid family isN-acetylneuraminic acid (Neu5Ac), which is the biosynthetic precursorfor most of the other types. Two major linkages between Neu5Ac and thepenultimate galactose residues of carbohydrate side chains are found innature, Neu5Ac α(2,3)-Gal and Neu5Ac α(2,6)-Gal. Both Neu5Ac α(2,3)-Galand Neu5Ac α(2,6)-Gal molecules can be recognized by influenza virusesand used as the receptor through which the virus binds and initiatesinfection. Human influenza viruses, however, seem to prefer Neu5Acα(2,6)-Gal, while avian and equine influenza viruses predominantlyrecognize Neu5Ac α(2,3)-Gal (Ito et al. (2000) Microobiol Immunol44:423-730). The human respiratory epithelium expresses both forms ofsialic acids, but α(2,6)-linked sialic acid is more abundant thanα(2,3)-linked sialic acid. The low abundance of α(2,3)-linked sialicacid is most likely the basis for the species barrier for avian viruses,and indicates that reducing the level of a receptor sialic acidexpressed on the airway epithelium would likely reduce the infectivityof an influenza virus. Thus, sialidases, which remove terminal sialicacid residues from sialo-glycoconjugates, present themselves aspotential influenza virus therapeutic agents that function to reduce thelevels of receptor sialic acids. Sialidases also can act as therapeuticagents for any other pathogen that utilizes sialic acids in theinfection process including, but not limited to, M. pneumoniae, M.catarrhalis, H. pylori, H. influenzae, S. pneumonia, P. aeruginosa,parainfluenza viruses and some coronaviruses and rotaviruses.

Sialidases tend to be highly substrate specific. They can targetparticular types of complex molecules, such as glycoproteins orglycolipids; specific sugar linkages (e.g. 2-3, 2-6, or 2-8); or can besensitive to the nature of the linkage sugar itself (e.g. D-galactose,N-acetyl-D-galactosamine). Substrate molecules include, but are notlimited to, oligosaccharides, polysaccharides, glycoproteins,gangliosides, and synthetic molecules. For example, a sialidase cancleave bonds having α(2,3)-Gal, α(2,6)-Gal, or α(2,8)-Gal linkagesbetween a sialic acid residue and the remainder of a substrate molecule.A sialidase also can cleave any or all of the linkages between thesialic acid residue and the remainder of the substrate molecule. Manysialidase proteins have been purified from microbes and highereukaryotes and of these, several have been shown to catalyze the removalof terminal sialic acid residues than can serve as receptors forpathogenic microorganisms. For example, among the large bacterialsialidases are those that that can degrade the influenza receptor sialicacids Neu5Ac α(2,6)-Gal and Neu5Ac α(2,3)-Gal, including sialidases fromClostridium perfringens, Actinomyces viscosus, Arthrobacter ureafaciens,and Micromonospora viridifaciens. Other sialidases that can serve astherapeutic agents include the human sialidases, such as those encodedby the genes NEU2 and NEU4.

Sialidase-GAG Fusion Proteins

Sialidase-GAG fusion proteins are proteins that are made up of asialidase protein, or catalytically active portion thereof, fused to aglycosaminoglycan (GAG)-binding sequence. As such, these proteinseffectively contain an anchoring domain (the GAG-binding sequence) and atherapeutic domain (the sialidase protein, or catalytically activeportion thereof). The sialidase-GAG fusion proteins are designed to bindto the epithelium and remove the surrounding sialic acids, and cantherefore be used as a therapeutic agent against pathogens that utilizesialic acids in the infection process. The ability of the fusion proteinto bind to the epithelium increases its retention when the fusionprotein is administered, for example, as an inhalant to treat influenzainfection. The GAG-binding sequence acts as an epithelium-anchoringdomain that tethers the sialidase to the respiratory epithelium andincreases its retention and potency.

Heparan sulfate, closely related to heparin, is a type ofglycosaminoglycan (GAG) that is ubiquitously present on cell membranes,including the surface of respiratory epithelium. Many proteinsspecifically bind to heparin/heparan sulfate, and the GAG-bindingsequences in these proteins have been identified. For example, theGAG-binding sequences of human platelet factor 4 (PF4) (SEQ ID NO:3),human interleukin 8 (IL8) (SEQ ID NO:4), human antithrombin III (AT III)(SEQ ID NO:5), human apoprotein E (ApoE) (SEQ ID NO:6), humanangio-associated migratory cell protein (AAMP) (SEQ ID NO:7), or humanamphiregulin (SEQ ID NO:8) have been shown to exhibit high affinity forheparin (Lee et al. (1991) PNAS 88:2768-2772; Goger et al. (2002)Biochem. 41:1640-1646; Witt et al. (1994) Curr Bio 4:394-400; Weisgraberet al. (1986) J Bio Chem 261:2068-2076). The GAG-binding sequences ofthese proteins are distinct from their receptor-binding sequences, sothey do not induce the biological activities associated with thefull-length proteins or the receptor-binding domains. These sequences,or other sequences that can bind heparin/heparan sulfate, can be used asepithelium-anchoring-domains in sialidase-GAG fusion proteins.

In the context of a sialidase-GAG fusion protein, the sialidase caninclude the entire sialidase protein, or a catalytically active portionthereof. For example, sialidase-GAG fusion protein can contain the 901amino acid sialidase protein from A. viscosus set forth in SEQ ID NO:1.In another example, the sialidase-GAG fusion protein can contain the 394amino acid catalytically active portion of a sialidase protein from A.viscosus set forth in SEQ ID NO:2. The GAG-binding sequence can belinked to the sialidase by recombinant methods. In some examples, thefusion protein can include an amino acid linker, such as four glycineresidues. Furthermore, linkage can be via the N- or C-terminus of theGAG-binding sequence, or the N- or C-terminus of the sialidase.Exemplary examples of sialidase-GAG fusion proteins include thosepolypeptides set forth in SEQ ID NOS: 9-13, and 17. In a furtherexample, the sialidase and GAG-binding sequence components can be linkedusing chemical or peptide linkers, by any method known in the art.

Proteinase Inhibitor 8

Proteinase inhibitor 8 (PI8), also known as Serpin B8, is a serineprotease inhibitor (serpin) Serpins are a large superfamily ofstructurally related proteins that are expressed in viruses, insects,plants and higher organisms, but not in bacteria or yeast. Serpinsregulate the activity of proteases involved in many biological process,including coagulation, fibrinolysis, inflammation, cell migration, andtumorigenesis. They contain a surface-exposed reactive site loop (RSL),which acts as a “bait” for proteases by mimicking a protease substratesequence. On binding of the target protease to the serpin, the RSL iscleaved, after which the protease is covalently linked to the serpin.The protease in the newly formed serpin-protease complex is inactive(Huntington et al. (2000) Nature 407:923-926).

PI8 is a member of a subfamily of serpins of which chicken ovalbumin isthe archtype. Like other serpins that belong to this family, PI8 lacks atypical cleavable N-terminal signal sequence, resulting in a 374 aminoacid protein (SEQ ID NO:14) that resides mainly intracellularly. Othermembers of this human ovalbumin-like subfamily include plasminogenactivator inhibitor type 2 (PAI-2), monocyte neutrophil elastaseinhibitor (MNEI), squamous cell carcinoma antigen (SCCA)-1, leupin(SCCA-2) maspin (PI5), protease inhibitor 6 (PI6), protease inhibitor(PI9) and bomapin (PI10). Within this family the serpins PI6, PI8, andPI9 show the highest structural homology (up to 68% amino acid identity)(Sprecher et al. (1995) J Biot Chem 270:29854-29861). PI-8 has beenshown to inhibit trypsin, thrombin, factor Xa, subtilisin A, furin, andalso chymotrypsin in vitro. It is released by platelets and appears tobe involved in the regulation of furin activity and, therefore, plateletaggregation (LeBlond et al. (2006) Thromb Haemost 95:243-252).

In addition to their role in the regulation of endogenous biologicalprocesses, such as coagulation, serine protease inhibitors also canfunction to inhibit the biological activities of exogenousmicroorganisms. For example, a number of serine protease inhibitors havebeen shown to reduce influenza virus activation in cultured cells,chicken embryos and in the lungs of infected mice. The serpins bind tohemagglutinin (HA) molecules on the surface of the influenza virus andinhibit its activity, thus reducing the infectivity of the virus. Forexample trypsin inhibitors, such as: aprotinin (Zhimov et al. (2002) JVirol 76:8682-8689), leupeptin (Zhimov et al. (2002) J Virol76:8682-8689; Tashiro et al. (1987) J Gen Virol 68:2039-2043), soybeanprotease inhibitor (Barbey-Morel et al. (1987) J Infect Dis155:667-672), e-aminocaproic acid (Zhimov et al. 1982. Arch Virol73:263-272) and n-p-tosyl-L-lysine chloromethylketone (TLCK)(Barbey-Morel et al. (1987) J Infect Dis 155:667-672) have all beenshown to inhibit influenza virus infection, and are candidatetherapeutic agents for use in the treatment of influenza virusinfection. Thus, as a related trypsin inhibitor, PI8 also can be used asa therapeutic agent in the treatment of influenza virus infection.

Surface Active Agents

The compositions provided herein can contain one or more surface activeagents that are added in an amount sufficient to form a stable emulsion.The appropriate amount of surface active agent is a function of thenon-denatured protein, optionally additional active agents for delivery,and other components present in the emulsion, since some agents can haveself-emulsifying properties and other agents and components affectsurface tension.

The surface active agents for use herein are substances which, whendissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous phase and theoil phase, to form a stable oil in water or water in oil emulsion. Thesurfactant molecules are amphiphilic and contain hydrophilic head groupsand hydrophobic tails. The surfactant molecules form variousmacro-molecular structure in an emulsion, such as micelles, inversemicelles, lipid bilayers (liposomes) and cubosomes. The exactmacromolecular structure which is formed depends on the relative sizesof the hydrophilic and hydrophobic regions of the surface activemolecule. In certain embodiments, the surface active agent is selectedfrom sodium lauryl sulfate; sorbitan laurate, sorbitan palmitate,sorbitan stearate (available under the tradename Span® 20-40-60 etc.);polysorbates such as polyoxyethylene (20) sorbitan monolaurate,polyoxyethylene (20) sorbitan monopalmitate, polyoxyethylene (20)sorbitan monostearate (available under the tradename TWEENS® 20-40-60etc.); benzalkonium chloride, mixed chain phospholipids, cationiclipids, oligolipids, phospholipids, carnitines, sphingosines,sphingomyelins, ceramides, glycolipids, lipoproteins, apoproteins,amphiphilic proteins, amphiphilic peptides, amphiphilic syntheticpolymers, and combinations thereof. Other exemplary surface activeagents for use herein include, but are not limited to

i) Natural lipids, i.e. Cholesterol, Sphingosine and Derivatives,Gangliosides, Sphingosine derivatives (Soy Bean), Phytosphingosine andderivatives (Yeast), Choline (Phosphatidylcholine), Ethanolamine(Phosphatidylethanolamine), Glycerol (Phosphatidyl-DL-glycerol),Inositol (Phosphatidylinositol), Serine (Phosphatidylserine (SodiumSalt)), Cardiolipin, Phosphatidic Acid, Egg Derived, Lyso (Mono Acyl)Derivatives (Lysophosphatides), Hydrogenated Phospholipids, Lipid TissueExtracts,

ii) Synthetic lipids, i.e. Asymmetric Fatty Acid, Symmetric FattyAcid—Saturated Series, Symmetric Fatty Acid—Unsaturated Series, AcylCoenzyme A (Acetoyl Coenzyme A, Butanoyl Coenzyme A, Crotanoyl CoenzymeA, Hexanoyl Coenzyme A, Octanoyl Coenzyme A, Decanoyl Coenzyme A,Lauroyl Coenzyme A, Myristoyl Coenzyme A, Palmitoyl Coenzyme A, StearoylCoenzyme A, Oleoyl Coenzyme A, Arachidoyl Coenzyme A, ArachidonoylCoenzyme A, Behenoyl Coenzyme A, Tricosanoyl Coenzyme A, LignoceroylCoenzyme A, Nervonoyl Coenzyme A, Hexacosanoyl Coenzyme A,

iii) Sphingolipids, i.e. D-erythro (C-18) Derivatives (Sphingosine, suchas: D-erythro Sphingosine (synthetic), Sphingosine-1-Phosphate, N,NDimethylsphingosine, N,N,N-Trimethylsphingosine,Sphingosylphosphorylcholine, Sphingomyelin and GlycosylatedSphingosine), Ceramide Derivatives (Ceramides, D-erythroCeramide-1-Phosphate, Glycosulated Ceramides), Sphinganine(Dihydrosphingosine) (Sphinganine-1-Phosphate, Sphinganine (C20),D-erythro Sphinganine, N-Acyl-Sphinganine C2, N-Acyl-Sphinganine C8,N-acyl-Sphinganine C16, N-Acyl-Sphinganine C18, N-Acyl-Sphinganine C24,N-Acyl-Sphinganine C24:1), Glycosylated (C18) Sphingosine andPhospholipid Derivatives (Glycosylated-Sphingosine) (Sphingosine, βD-Glucosyl, Sphingosine, β D-Galactosyl, Sphingosine,β D-Lactosyl),Glycosylated-Ceramide (D-Glucosyl-β1-1′ Ceramide (C8),D-Galactosyl-β1-1′ Ceramide (C8), D-Lactosyl-β1-1′ Ceramide (C8),D-Glucosyl-β1-1′ Ceramide (C12), D-Galactosyl-β1-1′ Ceramide (C12),D-Lactosyl-β1-1′ Ceramide (C12)), Glycosylated-Phosphatidylethanolamine(1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-Lactose), D-erythro(C17) Derivatives (D-erythro Sphingosine, D-erythroSphingosine-1-phosphate), D-erythro (C20) Derivatives (D-erythroSphingosine), L-threo (C18) Derivatives (L-threo Sphingosine, Safingol(L-threo Dihydrosphingosine)), Sphingosine Derivatives (Egg, Brain &Milk) (D-erythro-Sphingosine, Sphingomyelin, Ceramides, Cerebrosides,Brain Sulfatides), Gangliosides (Gangliosides Structures,Gangliosides—Ovine Brain, Gangliosides—Porcine Brain), SphingosineDerivatives (Soy Bean) (Glucosylceramide), Phytosphingosine Derivatives(Yeast) (Phytosphingosine, D-ribo-Phytosphingosine-1-Phosphate, N-AcylPhytosphingosine C2, N-Acyl Phytosphingosine C8, N-Acyl PhytosphingosineC18,

iv) Acyl coenzyme A, i.e. Acetoyl Coenzyme A (Ammonium Salt), ButanoylCoenzyme A (Ammonium Salt), Crotanoyl Coenzyme A (Ammonium Salt),Hexanoyl Coenzyme A (Ammonium Salt), Octanoyl Coenzyme A (AmmoniumSalt), Decanoyl Coenzyme A (Ammonium Salt), Lauroyl Coenzyme A (AmmoniumSalt), Myristoyl Coenzyme A (Ammonium Salt), Palmitoyl Coenzyme A(Ammonium Salt), Stearoyl Coenzyme A (Ammonium Salt), Oleoyl Coenzyme A(Ammonium Salt), Arachidoyl Coenzyme A (Ammonium Salt), ArachidonoylCoenzyme A (Ammonium Salt), Behenoyl Coenzyme A (Ammonium Salt),Tricosanoyl Coenzyme A (Ammonium Salt), Lignoceroyl Coenzyme A (AmmoniumSalt), Nervonoyl Coenzyme A (Ammonium Salt), Hexacosanoyl Coenzyme A(Ammonium Salt), Docosahexaenoyl Coenzyme A (Ammonium Salt),

v) Oxidized lipids, i.e.1-Palmitoyl-2-Azelaoyl-sn-Glycero-3-Phosphocholine,1-O-Hexadecyl-2-Azelaoyl-sn-Glycero-3-Phosphocholine,1-Palmitoyl-2-Glutaroyl-sn-Glycero-3-Phosphocholine (PGPC),1-Palmitoyl-2-(9′-oxo-Nonanoyl)-sn-Glycero-3-Phosphocholine,1-Palmitoyl-2-(5′-oxo-Valeroyl)-sn-Glycero-3-Phosphocholine,

vi) Ether lipids, i.e.: Diether Lipids (Dialkyl Phosphatidylcholine,Diphytanyl Ether Lipids), Alkyl Phosphocholine (Dodedylphosphocholine),O-Alkyl diacylphosphatidylcholinium(1,2-Diacyl-sn-Glycero-3-Ethylphosphocholine), Synthetic PAF &Derivatives (1-Alkyl-2-Acyl-Glycero-3-Phosphocholine & Derivatives),

vii) Fluorescent lipids, i.e.: Glycerol Based (Phosphatidylcholine(NBD), Phosphatidic Acid (NBD), Phosphatidylethanolamine (NBD),Phosphatidylglycerol (NBD), Phosphatidylserine (NBD)), Sphingosine Based(Ceramide (NBD), Sphingomyelin (NBD), Phytosphingosine (NBD), GalactosylCerebroside (NBD)), Headgroup Labeled Lipids (Glycerol Based)(Phosphatidylethanolamine (NBD), Phosphatidylethanolamine (LissamineRhodamine B), Dioleoyl Phosphatidylethanolamine (Dansyl, Pyrene,Fluorescein), Phosphatidylserine (NBD), Phosphatidylserine (Dansyl)),25-NBD-Cholesterol,

viii) Other lipids including, but not limited to Lecithin, Ultralec-P(ADM), Soy powder,

ix) Surfactants including, but not limited to polyethylene glycol 400;sodium lauryl sulfate; sorbitan laurate, sorbitan palmitate, sorbitanstearate (available under the tradename Span® 20-40-60 etc.);polysorbates such as polyoxyethylene (20) sorbitan monolaurate,polyoxyethylene (20) sorbitan monopalmitate, polyoxyethylene (20)sorbitan monostearate (available under the tradename TWEENS® 20-40-60etc.); benzalkonium chloride.

In certain embodiments, the phospholipids for use arephosphatidylcholines, phosphatidylethanolamines, phosphatidylserines,phosphatidylglycerols, phosphatidylinositols, phosphatidic acids, mixedchain phospholipids, lysophospholipids, hydrogenated phospholipids,partially hydrogenated phospholipids, and mixtures thereof.

In certain embodiments, the surface active agent is selected frompolysorbate-80, lecithin and phosphatidylcholine. The surface activeagents are present in an amount sufficient to form a stable emulsion.

The amount of surface active agent can be empirically determined and isa function of the agent selected, the desired form of the resultingcomposition. The amount include can be from less than 0.1% by weight upto 35% or more. In certain embodiments, the surface active agent ispresent at a concentration of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 15%, 20%, 25% by weight up to about 30% by weight of the totalweight of the composition. In certain embodiments, the surface activeagent is present at a concentration of about 1 weight % up to about 20weight % of the total weight of the composition. In certain embodiments,the surface active agent is present at a concentration of about 1 weight% up to about 15 weight % of the total weight of the composition. Inother embodiments, the surface active agent is present at aconcentration of about 1 weight % up to about 10 weight % of the totalweight of the composition. In other embodiments, the surface activeagent is present at a concentration of about 1 weight % up to about 8weight % of the total weight of the composition. In other embodiments,the surface active agent is present at a concentration of about 1 weight% up to about 6 weight % of the total weight of the composition. Inother embodiments, the surface active agent is present at aconcentration of about 1 weight % up to about 4 weight % of the totalweight of the composition. In other embodiments, the surface activeagent is present at a concentration of about 20 weight % of the totalweight of the composition. In other embodiments, the surface activeagent is present at a concentration of about 15 weight % of the totalweight of the composition. In other embodiments, the surface activeagent is present at a concentration of about 13 weight % of the totalweight of the composition. In other embodiments, the surface activeagent is present at a concentration of about 11 weight % of the totalweight of the composition. In other embodiments, the surface activeagent is present at a concentration of about 8 weight % of the totalweight of the composition. In other embodiments, the surface activeagent is present at a concentration of about 6 weight % of the totalweight of the composition. In other embodiments, the surface activeagent is present at a concentration of about 4 weight % of the totalweight of the composition. In other embodiments, the surface activeagent is present at a concentration of about 2 weight % of the totalweight of the composition. In other embodiments, the surface activeagent is present at a concentration of about 1 weight % of the totalweight of the composition.

The stable emulsions provided herein can contain one or more deliveryvehicles selected from among micelles, liposomes and cubosomes andmixtures thereof, or macromolecular assemblies of non-denatured proteinssuch as tubes, helices, spheres and the like, that can encapsulateadditional nutrients or active agents. The delivery vehiclesencapsulating the active agent are then absorbed in the epithelium wherethe non-denatured proteins and/or additional nutrients/active agents aredelivered.

Optional Additional Agents

The compositions provided herein can optionally, in addition tonon-denatured proteins, contain one or more pharmaceutical ornutraceutical or other such agent for ingestion by a subject. Generallythe agents are those that have a function in a host, e.g., immuneregulation, regulation of biochemical processes, or enzymatic activity.Any agent that can be formulated as described herein can be administeredin the compositions provided herein. Where the agent is a therapeuticthe compositions contain a therapeutically effective amount of an agentto be delivered. The particular amount of active agent in a dosage willvary widely according to the nature of the active agent, the nature ofthe condition being treated, the age and size of the subject, and otherparameters.

Generally, the amount of additional active agent or nutrient besides thenon-denatured proteins in the composition will vary from less than about0.01% by weight to about 20% by weight of the composition, or more andtypically are formulated for single dosage administration. A singledosage can vary from about 0.01 μg to 10 mg of an agent per kilogram ofbody weight of the host, with dosages from about 0.1 μg to 1 mg/kg beingcommonly employed. These concentrations, however, are general guidelinesonly and particular amounts and dosages may be selected based on theactive agent being administered, the condition being treated, and thetreatment regimen being employed means an amount of a drug or an activeagent that is sufficient to provide the desired local or systemic effectand performance at a reasonable benefit/risk ratio to a subjectattending any medical treatment.

Agents can be selected from inorganic and organic drugs including, butnot limited to drugs that act on the peripheral nerves, adrenergicreceptors, cholinergic receptors, nervous system, skeletal muscles,cardiovascular system, smooth muscles, blood circulatory system,synaptic sites, neuro-effector junctional sites, endocrine system,hormone systems, immunological system, reproductive system, skeletalsystem, autocoid systems, alimentary and excretory systems, histaminesystems, and the like. The active agents that can be delivered using thecompositions provided herein include, but are not limited to,anticonvulsants, analgesics, antiparkinsons, anti-inflammatories,calcium antagonists, anesthetics, antimicrobials, antimalarials,antiparasitics, antihypertensives, antihistamines, antipyretics,alpha-adrenergic agonists, alpha-blockers, biocides, bactericides,bronchial dilators, beta-adrenergic blocking drugs, contraceptives,cardiovascular drugs, calcium channel inhibitors, depressants,diagnostics, diuretics, electrolytes, enzymes, hypnotics, hormones,hypoglycemics, hyperglycemics, muscle contractants, muscle relaxants,neoplastics, glycoproteins, nucleoproteins, lipoproteins, ophthalmics,psychic energizers, sedatives, steroids, sympathomimetics,parasympathomimetics, tranquilizers, urinary tract drugs, vaccines,vaginal drugs, vitamins, minerals, nonsteroidal anti-inflammatory drugs,angiotensin converting enzymes, polynucleotides, polypeptides,polysaccharides, and nutritional supplements including herbalsupplements.

The level of agent to be delivered is from about 0.01% up to about 50%,from about 0.1% up to about 40%, from about 0.1% up to about 30%, fromabout 0.1% up to about 20%, from about 0.1% up to about 10%, from about0.1% up to about 9%, from about 0.1% up to about 8%, from about 0.1% upto about 7%, from about 0.1% up to about 6%, from about 0.1% up to about5%, from about 0.1% up to about 4%, from about 0.1% up to about 3%, fromabout 0.1% up to about 2%, from about 0.1% up to about 1% by weight ofthe composition. The agent to be delivered can be water soluble,slightly water soluble, or oil soluble. In certain embodiments, theagent to be delivered is selected from anticonvulsants, analgesics,antiparkinsons, anti-inflammatories, calcium antagonists, anesthetics,antimicrobials, antimalarials, antiparasitics, antihypertensives,antihistamines, antipyretics, alpha-adrenergic agonists, alpha-blockers,biocides, bactericides, bronchial dilators, beta-adrenergic blockingdrugs, contraceptives, cardiovascular drugs, calcium channel inhibitors,depressants, diagnostics, diuretics, electrolytes, enzymes, hypnotics,hormones, hypoglycemics, hyperglycemics, muscle contractants, musclerelaxants, neoplastics, glycoproteins, nucleoproteins, lipoproteins, nondenatured whey protein, ophthalmics, psychic energizers, sedatives,steroids, sympathomimetics, parasympathomimetics, tranquilizers, urinarytract drugs, vaccines, vaginal drugs, vitamins, minerals, nonsteroidalanti-inflammatory drugs, angiotensin converting enzymes,polynucleotides, polypeptides, polysaccharides, and nutritionalsupplements including herbal supplements.

In certain embodiments, the active agent is selected as follows:

α-Adrenergic agonists such as Adrafinil, Adrenolone, Amidephrine,Apraclonidine, Budralazine, Clonidine, Cyclopentamine, Detomidine,Dimetofrine, Dipivefrin, Ephedrine, Epinephrine, Fenoxazoline,Guanabenz, Guanfacine, Hydroxyamphetamine, Ibopamine, Indanazoline,Isometheptene, Mephentermine, Metaraminol, Methoxamine Hydrochloride,Methylhexaneamine, Metizolene, Midodrine, Naphazoline, Norepinephrine,Norfenefrine, Octodrine, Octopamine, Oxymetazoline, PhenylephrineHydrochloride, Phenylpropanolamine Hydrochloride,Phenylpropylmethylamine, Pholedrine, Propylhexedrine, Pseudoephedrine,Rilmenidine, Synephrine, Tetrahydrozoline, Tiamenidine, Tramazoline,Tuaminoheptane, Tymazoline, Tyramine and Xylometazoline;

β-Adrenergic agonists such as Albuterol, Bambuterol, Bitolterol,Carbuterol, Clenbuterol, Clorprenaline, Denopamine, Dioxethedrine,Dopexamine, Ephedrine, Epinephrine, Etafedrine, Ethylnorepinephrine,Fenoterol, Formoterol, Hexoprenaline, Ibopamine, Isoetharine,Isoproterenal, Mabuterol, Metaproterenol, Methoxyphenamine, Oxyfedrine,Pirbuterol, Prenalterol, Procaterol, Protokylol, Reproterol, Rimiterol,Ritodrine, Soterenol, Terbuterol and Xamoterol;

α-Adrenergic blockers such as Amosulalol, Arotinolol, Dapiprazole,Doxazosin, Ergoloid Mesylates, Fenspiride, Indoramin, Labetalol,Nicergoline, Prazosin, Terazosin, Tolazoline, Trimazosin and Yohimbine;

β-Adrenergic blockers such as Acebutolol, Alprenolol, Amosulalol,Arotinolol, Atenolol, Befunolol, Betaxolol, Bevantolol, Bisoprolol,Bopindolol, Bucumolol, Befetolol, Bufuralol, Bunitrolol, Bupranolol,Butidrine Hydrochloride, Butofilolol, Carazolol, Carteolol, Carvedilol,Celiprolol, Cetamolol, Cloranolol, Dilevalol, Epanolol, Esmolol,Indenolol, Labetalol, Levobunolol, Mepindolol, Metipranalol, Metoprolol,Moprolol, Nadoxolol, Nifenalol, Nipradilol, Oxprenolol, Penbutolol,Pindolol, Practolol, Pronethalol, Propranolol, Sotalol, Sulfinalol,Talinolol, Tertatolol, Timolol, Toliprolol and Xibenolol;

Alcohol deterrents such as Calcium Cyanamide Citrated, Disulfiram,Nadide and Nitrefazole;

Aldose reductase inhibitors such as Epalrestat, Ponalrestat, Sorbiniland Tolrestat;

Anabolics such as Androisoxazole, Androstenediol, Bolandiol,Bolasterone, Clostebol, Ethylestrenol; Formyldienolone,4-Hydroxy-19-nortestosterone, Methandriol, Methenolone,Methyltrienolone, Nandrolone, Nandrolone Decanoate, Nandrolonep-Hexyloxyphenylpropionate, Nandrolone Phenpropionate, Norbolethone,Oxymesterone, Pizotyline, Quinbolone, Stenbolone and Trenbolone;

Analgesics (dental) such as Chlorobutanol, Clove and Eugenol;

Analgesics (narcotic) such as Alfentanil, Allylprodine, Alphaprodine,Anileridine, Benzylmorphine, Bezitramide, Buprenorphine, Butorphanol,Clonitazene, Codeine, Codeine Methyl Bromide, Codeine Phosphate, CodeineSulfate, Desomorphine, Dextromoramide, Dezocine, Diampromide,Dihydrocodeine, Dihydrocodeinone Enol Acetate, Dihydromorphine,Dimenoxadol, Dimepheptanol, Dimethylthiambutene, Dioxaphetyl Butyrate,Dipipanone, Eptazocine, Ethoheptazine, Ethylmethlythiambutene,Ethylmorphine, Etonitazene, Fentanyl, Hydrocodone, HydrocodoneBitartrate, Hydromorphone, Hydroxypethidine, Isomethadone, Ketobemidone,Levorphanol, Lofentanil, Meperidine, Meptazinol, Metazocine, MethadoneHydrochloride, Metopon, Morphine, Morphine Derivatives, Myrophine,Nalbuphine, Narceine, Nicomorphine, Norlevorphanol, Normethadone,Normorphine, Norpipanone, Opium, Oxycodone, Oxymorphone, Papaveretum,Pentazocine, Phenadoxone, Phenazocine, Pheoperidine, Piminodine,Piritramide, Proheptazine, Promedol, Properidine, Propiram,Propoxyphene, Sufentanil and Tilidine;

Analgesics (non-narcotic) such as Acetaminophen, Acetaminosalol,Acetanilide, Acetylsalicylsalicylic Acid, Alclofenac, Alminoprofen,Aloxiprin, Aluminum Bis(acetylsalicylate), Aminochlorthenoxazin,2-Amino-4-picoline, Aminopropylon, Aminopyrine, Ammonium Salicylate,Antipyrine, Antipyrine Salicylate, Antrafenine, Apazone, Aspirin,Benorylate, Benoxaprofen, Benzpiperylon, Benzydamine,p-Bromoacetanilide, 5-Bromosalicylic Acid Acetate, Bucetin, Bufexamac,Bumadizon, Butacetin, Calcium Acetylsalicylate, Carbamazepine,Carbetidine, Carbiphene, Carsalam, Chloralantipyrine,Chlorthenoxazin(e), Choline Salicylate, Cinchophen, Ciramadol,Clometacin, Cropropamide, Crotethamide, Dexoxadrol, Difenamizole,Diflunisal, Dihydroxyaluminum Acetylsalicylate, Dipyrocetyl, Dipyrone,Emorfazone, Enfenamic Acid, Epirizole, Etersalate, Ethenzamide,Ethoxazene, Etodolac, Felbinac, Fenoprofen, Floctafenine, FlufenamicAcid, Fluoresone, Flupirtine, Fluproquazone, Flurbiprofen, Fosfosal,Gentisic Acid, Glafenine, Ibufenac, Imidazole Salicylate, Indomethacin,Indoprofen, Isofezolac, Isoladol, Isonixin, Ketoprofen, Ketorolac,p-Lactophenetide, Lefetamine, Loxoprofen, Lysine Acetylsalicylate,Magnesium Acetylsalicylate, Methotrimeprazine, Metofoline, Miroprofen,Morazone, Morpholine Salicylate, Naproxen, Nefopam, Nifenazone, 5′Nitro-2′ propoxyacetanilide, Parsalmide, Perisoxal, Phenacetin,Phenazopyridine Hydrochloride, Phenocoll, Phenopyrazone, PhenylAcetylsalicylate, Phenyl Salicylate, Phenyramidol, Pipebuzone,Piperylone, Prodilidine, Propacetamol, Propyphenazone, Proxazole,Quinine Salicylate, Ramifenazone, Rimazolium Metilsulfate, Salacetamide,Salicin, Salicylamide, Salicylamide O-Acetic Acid, Salicylsulfuric Acid,Salsalte, Salverine, Simetride, Sodium Salicylate, Sulfamipyrine,Suprofen, Talniflumate, Tenoxicam, Terofenamate, Tetradrine, Tinoridine,Tolfenamic Acid, Tolpronine, Tramadol, Viminol, Xenbucin and Zomepirac;

Androgens such as Androsterone, Boldenone, Dehydroepiandrosterone,Fluoxymesterone, Mestanolone, Mesterolone, Methandrostenolone,17-Methyltestosterone, 17α-Methyltestosterone 3-Cyclopentyl Enol Ether,Norethandrolone, Normethandrone, Oxandrolone, Oxymesterone,Oxymetholone, Prasterone, Stanlolone, Stanozolol, Testosterone,Testosterone 17-Chloral Hemiacetal, Testosterone 17β-Cypionate,Testosterone Enanthate, Testosterone Nicotinate, TestosteronePheynylacetate, Testosterone Propionate and Tiomesterone;

Anesthetics such as Acetamidoeugenol, Alfadolone Acetate, Alfaxalone,Amucaine, Amolanone, Amylocaine Hydrochloride, Benoxinate, Benzocaine,Betoxycaine, Biphenamine, Bupivacaine, Butacaine, Butaben,Butanilicaine, Burethamine, Buthalital Sodium, Butoxycaine, Carticaine,2-Chloroprocaine Hydrochloride, Cocaethylene, Cocaine, Cyclomethycaine,Dibucaine Hydrochloride, Dimethisoquin, Dimethocaine, DiperadonHydrochloride, Dyclonine, Ecgonidine, Ecgonine, Ethyl Aminobenzoate,Ethyl Chloride, Etidocaine, Etoxadrol, β-Eucaine, Euprocin, Fenalcomine,Fomocaine, Hexobarbital, Hexylcaine Hydrochloride, Hydroxydione Sodium,Hydroxyprocaine, Hydroxytetracaine, Isobutyl p-Aminobenzoate, Kentamine,Leucinocaine Mesylate, Levoxadrol, Lidocaine, Mepivacaine, MeprylcaineHydrochloride, Metabutoxycaine Hydrochloride, Methohexital Sodium,Methyl Chloride, Midazolam, Myrtecaine, Naepaine, Octacaine, Orthocaine,Oxethazaine, Parethoxycaine, Phenacaine Hydrochloride, Phencyclidine,Phenol, Piperocaine, Piridocaine, Polidocanol, Pramoxine, Prilocaine,Procaine, Propanidid, Propanocaine, Proparacaine, Propipocaine,Propofol, Propoxycaine Hydrochloride, Pseudococaine, Pyrrocaine, QuinineUrea Hydochloride, Risocaine, Salicyl Alcohol, Tetracaine Hydrochloride,Thialbarbital, Thimylal, Thiobutabarbital, Thiopental Sodium, Tolycaine,Trimecaine and Zolamine;

Anorexics such as Aminorex, Amphecloral, Amphetamine, Benzaphetamine,Chlorphentermine, Clobenzorex, Cloforex, Clortermine, Cyclexedrine,Destroamphetamine Sulfate, Diethylpropion, Diphemethoxidine,N-Ethylamphetamine, Fenbutrazate, Fenfluramine, Fenproporex,Furfurylmethylamphetamine, Levophacetoperate, Mazindol, Mefenorex,Metamfeproamone, Methamphetamine, Norpseudoephedrine, Phendimetrazine,Phendimetrazine Tartrate, Phenmetrazine, Phenpentermine,Phenylpropanolamine Hydrochloride and Picilorex;

Anthelmintics (Cestodes) such as Arecoline, Aspidin, Aspidinol,Dichlorophen(e), Embelin, Kosin, Napthalene, Niclosamide, Pellertierine,Pellertierine Tannate and Quinacrine;

Anthelmintics (Nematodes) such as Alantolactone, Amoscanate, Ascaridole,Bephenium, Bitoscanate, Carbon Tetrachloride, Carvacrol, Cyclobendazole,Diethylcarbamazine, Diphenane, Dithiazanine Iodide, Dymanthine, GentianViolet, 4-Hexylresorcinol, Kainic Acid, Mebendazole, 2-Napthol, Oxantel,Papain, Piperazine, Piperazine Adipate, Piperazine Citrate, PiperazineEdetate Calcium, Piperazine Tartrate, Pyrantel, Pyrvinium Pamoate,α-Santonin, Stilbazium Iodide, Tetrachloroethylene, Tetramisole,thiabendazole, Thymol, Thymyl N-Isoamylcarbamate, Triclofenol Piperazineand Urea Stibamine;

Anthelmintics (Onchocerca) such as Ivermectin and Suramin Sodium;

Anthelmintics (Schistosoma) such as Amoscanate, Amphotalide, AntimonyPotassium Tartrate, Antimony Sodium Gluconate, Antimony Sodium Tartrate,Antimony Sodium Thioglycollate, Antimony Thioglycollamide, Becanthone,Hycanthone, Lucanthone Hydrochloride, Niridazole, Oxamniquine,Praziquantel, Stibocaptate, Stibophen and Urea Stibamine;

Anthelmintic (Trematodes) such as Anthiolimine and Tetrachloroethylene;

Antiacne drugs such as Adapelene, Algestone Acetophenide, Azelaic Acid,Benzoyl Peroxide, Cyoctol, Cyproterone, Motretinide, Resorcinol,Retinoic Acid, Tetroquinone and Tretinonine;

Antiallergics such as Amlexanox, Astemizole, Azelastine, Cromolyn,Fenpiprane, Histamine, Ibudilast, Nedocromil, Oxatomide, Pentigetide,Poison Ivy Extract, Poison Oak Extract, Poison Sumac Extract,Repirinast, Tranilast, Traxanox and Urushiol;

Antiamebics such as Arsthinol, Bialamicol, Carbarsone, Cephaeline,Chlorbetamide, Chloroquine, Chlorphenoxamide, Chlortetracycline,Dehydroemetine, Dibromopropamidine, Diloxanide, Dephetarsone, Emetine,Fumagillin, Glaucarubin, Glycobiarsol,8-Hydroxy-7-iodo-5-quinolinesulfonic Acid, Iodochlorhydroxyquin,Iodoquinol, Paromomycin, Phanquinone, Phearsone Sulfoxylate,Polybenzarsol, Propamidine, Quinfamide, Secnidazole, Sulfarside,Teclozan, Tetracycline, Thiocarbamizine, Thiocarbarsone and Tinidazole;

Antiandrogens such as Bifluranol, Cyoctol, Cyproterone, DelmadinoneAcetate, Flutimide, Nilutamide and Oxendolone;

Antianginals such as Acebutolol, Alprenolol, Amiodarone, Amlodipine,Arotinolol, Atenolol, Bepridil, Bevantolol, Bucumolol, Bufetolol,Bufuralol, Bunitrolol, Bupranolol, Carozolol, Carteolol, Carvedilol,Celiprolol, Cinepazet Maleate, Diltiazem, Epanolol, Felodipine,Gallopamil, Imolamine, Indenolol, Isosorbide Dinitrate, Isradipine,Limaprost, Mepindolol, Metoprolol, Molsidomine, Nadolol, Nicardipine,Nifedipine, Nifenalol, Nilvadipine, Nipradilol, Nisoldipine,Nitroglycerin, Oxprenolol, Oxyfedrine, Ozagrel, Penbutolol,Pentaerythritol Tetranitrate, Pindolol, Pronethalol, Propranolol,Sotalol, Terodiline, Timolol, Toliprolol and Verapamil;

Antiarrhythmics such as Acebutol, Acecaine, Adenosine, Ajmaline,Alprenolol, Amiodarone, Amoproxan, Aprindine, Arotinolol, Atenolol,Bevantolol, Bretylium Tosylate, Bubumolol, Bufetolol, Bunaftine,Bunitrolol, Bupranolol, Butidrine Hydrochloride, Butobendine, CapobenicAcid, Carazolol, Carteolol, Cifenline, Cloranolol, Disopyramide,Encainide, Esmolol, Flecainide, Gallopamil, Hydroquinidine, Indecainide,Indenolol, Ipratropium Bromide, Lidocaine, Lorajmine, Lorcainide,Meobentine, Metipranolol, Mexiletine, Moricizine, Nadoxolol, Nifenalol,Oxprenolol, Penbutolol, Pindolol, Pirmenol, Practolol, Prajmaline,Procainamide Hydrochloride, Pronethalol, Propafenone, Propranolol,Pyrinoline, Quinidine Sulfate, Quinidine, Sotalol, Talinolol, Timolol,Tocainide, Verapamil, Viquidil and Xibenolol; Antiarteriosclerotics suchas Pyridinol Carbamate;

Antiarthritic/Antirheumatics such as Allocupreide Sodium, Auranofin,

Aurothioglucose, Aurothioglycanide, Azathioprine, Calcium3-Aurothio-2-propanol-1-sulfonate, Celecoxib, Chloroquine, Clobuzarit,Cuproxoline, Diacerein, Glucosamine, Gold Sodium Thiomalate, Gold SodiumThiosulfate, Hydroxychloroquine, Kebuzone, Lobenzarit, Melittin,Methotrexate, Myoral and Penicillamine;

Antibacterial (antibiotic) drugs including: Aminoglycosides such asAmikacin, Apramycin, Arbekacin, Bambermycins, Butirosin, Dibekacin,Dihdrostreptomycin, Fortimicin(s), Gentamicin, Ispamicin, Kanamycin,Micronomicin, Neomycin, Neomycin Undecylenate, Netilmicin, Paromomycin,Ribostamycin, Sisomicin, Spectinomycin, Streptomycin, Streptonicozid andTobramycin;

Amphenicols such as Azidamfenicol, Chloramphenicol, ChloramphenicolPalmitate, Chloramphenicol Pantothenate, Florfenicol and Thiamphenicol;

Ansamycins such as Rifamide, Rifampin, Rifamycin and Rifaximin;

β-Lactams, including: Carbapenems such as Imipenem;

Cephalosporins such as Cefactor, Cefadroxil, Cefamandole, Cefatrizine,Cefazedone, Cefazolin, Cefixime, Cefmenoxime, Cefodizime, Cefonicid,Cefoperazone, Ceforanide, Cefotaxime, Cefotiam, Cefpimizole,Cefpirimide, Cefpodoxime Proxetil, Cefroxadine, Cefsulodin, Ceftazidime,Cefteram, Ceftezole, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefuroxime,Cefuzonam, Cephacetrile Sodium, Cephalexin, Cephaloglycin,Cephaloridine, Cephalosporin, Cephalothin, Cephapirin Sodium, Cephradineand Pivcefalexin;

Cephamycins such as Cefbuperazone, Cefmetazole, Cefminox, Cefetan andCefoxitin;

Monobactams such as Aztreonam, Carumonam and Tigemonam;

Oxacephems such as Flomoxef and Moxolactam;

Penicillins such as Amidinocillin, Amdinocillin Pivoxil, Amoxicillin,Ampicillan, Apalcillin, Aspoxicillin, Azidocillan, Azlocillan,Bacampicillin, Benzylpenicillinic Acid, Benzylpenicillin Sodium,Carbenicillin, Carfecillin Sodium, Carindacillin, Clometocill in,Cloxacill in, Cyclacillin, Dicloxacillin, Diphenicillin Sodium,Epicillin, Fenbenicillin, Floxicillin, Hetacillin, Lenampicillin,Metampicillin, Methicillin Sodium, Mezlocillin, Nafcillin Sodium,Oxacillin, Penamecillin, Penethamate Hydriodide, Penicillin GBenethamine, Penicillin G Benzathine, Penicillin G Benzhydrylamine,Penicillin G Calcium, Penicillin G Hydrabamine, Penicillin G Potassium,Penicillin G Procaine, Penicillen N, Penicillin O, Penicillin V,Penicillin V Benzathine, Penicillin V Hydrabamine, Penimepicycline,Phenethicillin Potassium, Piperacillin, Pivapicillin, Propicillin,Quinacillin, Sulbenicillin, Talampicillin, Temocillin and Ticarcillin;

Lincosamides such as Clindamycin and Lincomycin;

Macrolides such as Azithroimycin, Carbomycin, Clarithromycin,Erythromycin, Erythromycin Acistrate, Erythromycin Estolate,Erythromycin Glucoheptonate, Erythromycin Lactobionate, ErythromycinPropionate, Erythromycin Stearate, Josamycin, Leucomycins, Midecamycins,Miokamycin, Oleandomycin, Primycin, Rokitamycin, Rosaramicin,Roxithromycin, Spiramycin and Troleandomycin;

Polypeptides such as Amphomycin, Bacitracin, Capreomycin, Colistin,Enduracidin, Enviomycin, Fusafungine, Gramicidin(s), Gramicidin S,Mikamycin, Polymyxin, Polymyxin B-Methanesulfonic Acid, Pristinamycin,Ristocetin, Teicoplanin, Thiostrepton, Tuberactinomycin, Tyrocidine,Tyrothricin, Vancomycin, Viomycin, Viomycin Pantothenate, Virginiamycinand Zinc Bacitracin;

Tetracyclines such as Apicycline, Chlortetracycline, Clomocycline,Demeclocycline, Doxycycline, Guamecycline, Lymecycline, Meclocycline,Methacycline, Minocycline, Oxytetracycline, Penimepicycline,Pipacycline, Rolitetracycline, Sancycline, Senociclin and Tetracycline;and

other antibiotics such as Cycloserine, Mupirocin and Tuberin;

Antibacterial drugs (synthetic), including: 2,4-Diaminopyrimidines suchas Brodimoprim, Tetroxoprim and Trimethoprim;

Nitrofurans such as Furaltadone, Furazolium Chloride, Nifuradene,Nifuratel, Nifurfoline, Nifurpirinol, Nifurprazine, Nifurtoinol andNitrofurantoin;

Quinolones and Analogs such as Amifloxacin, Cinoxacin, Ciprofloxacin,Difloxacin, Enoxacin, Fleroxacin, Flumequine, Lomefloxacin, Miloxacin,Nalidixic Acid, Norfloxacin, Ofloxacin, Oxolinic Acid, Pefloxacin,Pipemidic Acid, Piromidic Acid, Rosoxacin, Temafloxacin andTosufloxacin;

Sulfonamides such as Acetyl Sulfamethoxypyrazine, Acetyl Sulfisoxazole,Azosulfamide, Benzylsulfamide, Chloramine-B, Chloramine-T, DichloramineT, Formosulfathiazole, N₂ Formylsulfisomidine,N²-β-D-Glucosylsulfanilamide, Mafenide,4′-(Methylsulfamoyl)sulfanilanilide, p-Nitrosulfathiazole,Noprylsulfamide, Phthalylsulfacetamide, Phthalylsulfathiazole,Salazosulfadimidine, Succinylsulfathiazole, Sulfabenzamide,Sulfacetamide, Sulfachlorpyridazine, Sulfachrysoidine, Sulfacytine,Sulfadiazine, Sulfadicramide, Sulfadimethoxine, Sulfadoxine,Sulfaethidole, Sulfaguanidine, Sulfaguanol, Sulfalene, Sulfaloxic Acid,Sulfamerazine, Sulfameter, Sulfamethazine, Sulfamethizole,Sulfamethomidine, Sulfamethoxazole, Sulfamethoxypyridazine,Sulfametrole, Sulfamidochrysoidine, Sulfamoxole, Sulfanilamide,Sulfanilamidomethanesulfonic Acid Triethanolamine Salt,4-Sulfanilamidosalicylic Acid, N-Sulfanilylsulfanilamide,Sulfanilylurea, N-Sulfanilyl-3,4-xylamide, Sulfanitran, Sulfaperine,Sulfaphenazole, Sulfaproxyline, Sulfapyrazine, Sulfapyridine,Sulfasomizole, Sulfasymazine, Sulfathiazole, Sulfathiourea,Sulfatolamide, Sulfisomidine and Sulfisoxazole;

Sulfones such as Acedapsone, Acediasulfone, Acetosulfone Sodium,Dapsone, Diathymosulfone, Glucosulfone Sodium, Solasulfone,Succisulfone, Sulfanilic Acid, p-Sulfanilylbenzylamine,p,p′-Sulfonyldianiline-N,N′digalactoside, Sulfoxone Sodium andThiazolsulfone; and

others such as Clofoctol, Hexedine, Methenamine, MethenamineAnhydromethylene-citrate, Methenamine Hippurate, Methenamine Mandelate,Methenamine Sulfosalicylate, Nitroxoline and Xibornol;

Anticholinergics such as Adiphenine Hydrochloride, Alverine,Ambutonomium Bromide, Aminopentamide, Amixetrine, AmprotropinePhosphate, Anisotropine Methylbromide, Apoatropine, Atropine, AtropineN-Oxide, Benactyzine, Benapryzine, Benzetimide, Benzilonium Bromide,Benztropine Mesylate, Bevonium Methyl Sulfate, Biperiden, ButropiumBromide, N-Butylscopolammonium Bromide, Buzepide, Camylofine, CaramiphenHydrochloride, Chlorbenzoxamine, Chlorphenoxamine, Cimetropium Bromide,Clidinium Bromide, Cyclodrine, Cyclonium Iodide, CycrimineHydrochloride, Deptropine, Dexetimide, Dibutoline Sulfate, DicyclomineHydrochloride, Diethazine, Difemerine, Dihexyverine, DiphemanilMethylsulfate, N-(1,2-Diphenylethyl) nicotinamide, Dipiproverine,Diponium Bromide, Emepronium Bromide, Endobenzyline Bromide,Ethopropazine, Ethybenztropine, Ethylbenzhydramine, Etomidoline,Eucatropine, Fenpiverinium Bromide, Fentonium Bromide, FlutropiumBromide, Glycopyrrolate, Heteronium Bromide, Hexocyclium Methyl Sulfate,Homatropine, Hyoscyamine, Ipratropium Bromide, Isopropamide, Levomepate,Mecloxamine, Mepenzolate Bromide, Metcaraphen, Methantheline Bromide,Methixene, Methscopolamine Bromide, Octamylamine, Oxybutynin Chloride,Oxyphencyclimine, Oxyphenonium Bromide, Pentapiperide, PenthienateBromide, Phencarbamide, Phenglutarimide, Pipenzolate Bromide,Piperidolate, Piperilate, Poldine Methysulfate, Pridinol, PrifiniumBromide, Procyclidine, Propantheline Bromide, Propenzolate,Propyromazine, Scopolamine, Scopolamine N-Oxide, Stilonium Iodide,Stramonium, Sultroponium, Thihexinol, Thiphenamil, Tiemonium Iodide,Timepidium Bromide, Tiquizium Bromide, Tridihexethyl Iodide,Trihexyphenidyl Hydrochloride, Tropacine, Tropenzile, Tropicamide,Trospium Chloride, Valethamate Bromide and Xenytropium Bromide;

Anticonvulsants such as Acetylpheneturide, Albutoin, Aloxidone,Aminoglutethimide, 4-Amino-3-hydroxybutyric Acid, Atrolactamide,Beclamide, Buramate, Calcium Bromide, Carbamazepine, Cinromide,Clomethiazole, Clonazepam, Decimemide, Diethadione, Dimethadione,Doxenitoin, Eterobarb, Ethadione, Ethosuximide, Ethotoin, Fluoresone,Garbapentin, 5-Hydroxytryptophan, Lamotrigine, Lomactil, MagnesiumBromide, Magnesium Sulfate, Mephenytoin, Mephobarbital, Metharbital,Methetoin, Methsuximide, 5-Methyl-5-(3-phenanthryl)hydantoin,3-Methyl-5-phenylhydantoin, Narcobarbital, Nimetazepam, Nitrazepam,Paramethadione, Phenacemide, Phenetharbital, Pheneturide, Phenobarbital,Phenobarbital Sodium, Phensuximide, Phenylmethylbarbituric Acid,Phenytoin, Phethenylate Sodium, Potassium Bromide, Pregabatin,Primidone, Progabide, Sodium Bromide, Sodium Valproate, Solanum,Strontium Bromide, Suclofenide, Sulthiame, Tetrantoin, Tiagabine,Trimethadione, Valproic Acid, Valpromide, Vigabatrin and Zonisamide;

Antidepressants, including: Bicyclics such as Binedaline, Caroxazone,Citalopram, Dimethazan, Indalpine, Fencamine, Fluvoxamine Maleate,Indeloxazine Hydrochcloride, Nefopam, Nomifensine, Oxitriptan,Oxypertine, Paroxetine, Sertraline, Thiazesim, Trazodone, Venlafaxineand Zometapine;

Hydrazides/Hydrazines such as Benmoxine, Iproclozide, Iproniazid,Isocarboxazid, Nialamide, Octamoxin and Phenelzine;

Pyrrolidones such as Cotinine, Rolicyprine and Rolipram;

Tetracyclics such as Maprotiline, Metralindole, Mianserin andOxaprotiline;

Tricyclics such as Adinazolam, Amitriptyline, Amitriptylinoxide,Amoxapine, Butriptyline, Clomipramine, Demexiptiline, Desipramine,Dibenzepin, Dimetracrine, Dothiepin, Doxepin, Fluacizine, Imipramine,Imipramine N-Oxide, Iprindole, Lofepramine, Melitracen, Metapramine,Nortriptyline, Noxiptilin, Opipramol, Pizotyline, Propizepine,Protriptyline, Quinupramine, Tianeptine and Trimipramine; and

others such as Adrafinil, Benactyzine, Bupropion, Butacetin, Deanol,Deanol Aceglumate, Deanol Acetamidobenzoate, Dioxadrol, Etoperidone,Febarbamate, Femoxetine, Fenpentadiol, Fluoxetine, Fluvoxamine,Hematoporphyrin, Hypercinin, Levophacetoperane, Medifoxamine, Minaprine,Moclobemide, Oxaflozane, Piberaline, Prolintane, Pyrisuccideanol,Rubidium Chloride, Sulpiride, Sultopride, Teniloxazine, Thozalinone,Tofenacin, Toloxatone, Tranylcypromine, L-Tryptophan, Viloxazine andZimeldine;

Antidiabetics, including: Biguanides such as Buformin, Metformin andPhenformin;

Hormones such as Glucagon, Insulin, Insulin Injection, Insulin ZincSuspension, Isophane Insulin Suspension, Protamine Zinc InsulinSuspension and Zinc Insulin Crystals;

Sulfonylurea derivatives such as Acetohexamide, 1-Butyl-3-metanilylurea,Carbutamide, Chlorpropamide, Glibornuride, Gliclazide, Glipizide,Gliquidone, Glisoxepid, Glyburide, Glybuthiazol(e), Glybuzole,Glyhexamide, Glymidine, Glypinamide, Phenbutamide, Tolazamide,Tolbutamide and Tolcyclamide; and

others such as Acarbose, Calcium Mesoxalate and Miglitol;

Antidiarrheal drugs such as Acetyltannic Acid, Albumin Tannate,Alkofanone, Aluminum Salicylates—Basic, Catechin, Difenoxin,Diphenoxylate, Lidamidine, Loperamide, Mebiquine, Trillium and Uzarin;

Antidiuretics such as Desmopressin, Felypressin, Lypressin, Ornipressin,Oxycinchophen, Pituitary—Posterior, Terlipressin and Vasopressin;

Antiestrogens such as Delmadinone Acetate, Ethamoxytriphetol, Tamoxifenand Toremifene;

Antifungal drugs (antibiotics), including: Polyenes such asAmphotericin-B, Candicidin, Dermostatin, Filipin, Fungichromin,Hachimycin, Hamycin, Lucensomycin, Mepartricin, Natamycin, Nystatin,Pecilocin and Perimycin; and others such as Azaserine, Griseofulvin,Oligomycins, Neomycin Undecylenate, Pyrrolnitrin, Siccanin, Tubercidinand Viridin;

Antifungal drugs (synthetic), including: Allylamines such as Naftifineand Terbinafine;

Imidazoles such as Bifonazole, Butoconazole, Chlordantoin,Chlormidazole, Cloconazole, Clotrimazole, Econazole, Enilconazole,Fenticonazole, Isoconazole, Ketoconazole, Miconazole, Omoconazole,Oxiconazole, Nitrate, Sulconazole and Tioconazole;

Triazoles such as Fluconazole, Itraconazole and Terconazole; and

others such as Acrisorcin, Amorolfine, Biphenamine,Bromosalicylchloranilide, Buclosamide, Calcium Propionate, Chlophenesin,Ciclopirox, Cloxyquin, Coparaffinate, Diamthazole, Dihydrochloride,Exalamide, Flucytosine, Halethazole, Hexetidine, Loflucarban, Nifuratel,Potassium Iodide, Propionic Acid, Pyrithione, Salicylanilide, SodiumPropionate, Sulbentine, Tenonitrozole, Tolciclate, Tolindate,Tolnaftate, Tricetin, Ujothion, Undecylenic Acid and Zinc Propionate;

Antiglaucoma drugs such as Acetazolamide, Befunolol, Betaxolol,Bupranolol, Carteolol, Dapiprazoke, Dichlorphenamide, Dipivefrin,Epinephrine, Levobunolol, Methazolamide, Metipranolol, Pilocarpine,Pindolol and Timolol;

Antigonadotropins such as Danazol, Gestrinone and Paroxypropione;

Antigout drugs such as Allopurinol, Carprofen, Colchicine, Probenecidand Sulfinpyrazone;

Antihistamines, including: Alkylamine derivatives such as Acrivastine,Bamipine, Brompheniramine, Chlorpheniramine, Dimethindene, Metron S,Pheniramine, Pyrrobutamine, Thenaldine, Tolpropamine and Triprolidine;

Aminoalkyl ethers such as Bietanautine, Bromodiphenhydramine,Carbinoxamine, Clemastine, Diphenlypyraline, Doxylamine, Embrammine,Medrylamine, Mephenphydramine, p-Methyldiphenhydramine, Orphenadrine,Phenyltoloxamine, Piprinhydrinate and Setasine;

Ethylenediamine derivatives such as Alloclamide, p-Bromtripelennamine,Chloropyramine, Chlorothen, Histapyrrodine, Methafurylene,Methaphenilene, Methapyrilene, Phenbenzamine, Pyrilamine, Talastine,Thenyldiamine, Thonzylamine Hydrochloride, Tripelennamine and Zolamine;

Piperazines such as Cetirizine, Chlorcyclizine, Cinnarizine, Clocinizineand Hydroxyzine;

Tricyclics, including: Phenothiazines such as Ahistan, Etymemazine,Fenethazine, N-Hydroxyethylpromethazine Chloride, Isopromethazine,Mequitazine, Promethazine, Pyrathiazine and Thiazinamium Methyl Sulfate;and

others such as Azatadine, Clobenzepam, Cyproheptadine, Deptropine,Isothipendyl, Loratadine and Prothipendyl; and

other antihistamines such as Antazoline, Astemizole, Azelastine,Cetoxime, Clemizole, Clobenztropine, Diphenazoline, Diphenhydramine,Fluticasone Propionate, Mebhydroline, Phenindamine, Terfenadine andTritoqualine;

Antihyperlipoproteinemics, including: Aryloxyalkanoic acid derivativessuch as Beclorbrate, Bazafibrate, Binifibrate, Ciprofibrate,Clinofibrate, Clofibrate, Clofibric Acid, Etonfibrate, Fenofibrate,Gemfibrozil, Nicofibrate, Pirifibrate, Ronifibrate, Simfibrate andTheofibrate;

Bile acid sequesterants such as Cholestyramine Resin, Colestipol andPolidexide;

HMG CoA reductase inhibitors such as Fluvastatin, Lovastatin,Pravastatin Sodium and Simvastatin;

Nicotinic acid derivatives Aluminum Nicotinate, Acipimox, Niceritrol,Nicoclonate, Nicomol and Oxiniacic Acid;

Thyroid hormones and analogs such as Etiroxate, Thyropropic Acid andThyroxine; and

others such as Acifran, Azacosterol, Benfluorex, β-Benzalbutyramide,Carnitine, Chondroitin Sulfate, Clomestone, Detaxtran, Dextran SulfateSodium, 5,8,11,14,17-Eicosapentaenoic Acid, Eritadenine, Furazbol,Meglutol, Melinamide, Mytatrienediol, Ornithine, γ-Oryzanol, Pantethine,Penataerythritol Tetraacetate, α-Phenylbutyramide, Pirozadil, Probucol,α-Sitosterol, Sultosilic Acid, Piperazine Salt, Tiadenol, Triparanol andXenbucin;

Antihypertensive drugs, including: Arylethanolamine derivatives such asAmosulalol, Bufuralol, Dilevalol, Labetalol, Pronethalol, Sotalol andSulfinalol;

Aryloxypropanolamine derivatives such as Acebutolol, Alprenolol,Arotinolol, Atenolol, Betaxolol, Bevantolol, Bisoprolol, Bopindolol,Bunitrolol, Bupranolol, Butofilolol, Carazolol, Cartezolol, Carvedilol,Celiprolol, Cetamolol, Epanolol, Indenolol, Mepindolol, Metipranolol,Metoprolol, Moprolol, Nadolol, Nipradilol, Oxprenolol, Penbutolol,Pindolol, Propranolol, Talinolol, Tetraolol, Timolol and Toliprolol;

Benzothiadiazine derivatives such as Althiazide, Bendroflumethiazide,Benzthiazide, Benzylhydrochlorothiazide, Buthiazide, Chlorothiazide,Chlorthalidone, Cyclopenthiazide, Cyclothiazide, Diazoxide, Epithiazide,Ethiazide, Fenquizone, Hydrochlorothiazide, Hydroflumethiazide,Methyclothiazide, Meticrane, Metolazone, Paraflutizide, Polythiazide,Tetrachlormethiazide and Trichlormethiazide;

N-Carboxyalkyl (peptide/lactam) derivatives such as Alacepril,Captopril, Cilazapril, Delapril, Enalapril, Enalaprilat, Fosinopril,Lisinopril, Moveltipril, Perindopril, Quinapril and Ramipril;

Dihydropyridine derivatives such as Amlodipine, Felodipine, Isradipine,Nicardipine, Nifedipine, Nilvadipine, Nisoldipine and Nitrendipirne;

Guanidine derivatives such as Bethanidine, Debrisoquin, Guanabenz,Guanacline, Guanadrel, Guanazodine, Guanethidine, Guanfacine,Guanochlor, Guanoxabenz and Guanoxan;

Hydrazines and phthalazines such as Budralazine, Cadralazine,Dihydralazine, Endralazine, Hydracarbazine, Hydralazine, Pheniprazine,Pildralazine and Todralazine;

Imidazole derivatives such as Clonidine, Lofexidine, Phentolamine,Phentolamine Mesylate, Tiamenidine and Tolonidine;

Quaternary ammonium compounds Azamethonium Bromide, ChlorisondamineChloride, Hexamethonium, Pentacynium Bis(methyl sulfate), PentamethoniumBromide, Pentolinium Tartate, Phenactopinium Chloride and TrimethidiunumMethosulfate;

Quinazoline derivatives such as Alfuzosin, Bunazosin, Doxazosin,Prasosin, Terazosin and Trimazosin;

Reserpine derivatives such as Bietaserpine, Deserpidine, Rescinnamine,Reserpine and Syrosingopine;

Sulfonamide derivatives such as Ambuside, Clopamide, Furosemide,Indapamide, Quinethazone, Tripamide and Xipamide; and

others such as Ajmaline, γ-Aminobutyric Acid, Bufeniode, Candesartan,Chlorthalidone, Cicletaine, Ciclosidomine, Cryptenamine Tannates,Eprosartan, Fenoldopam, Flosequinan, Indoramin, Irbesartan, Ketanserin,Losartan, Metbutamate, Mecamylamine, Methyldopa, Methyl 4-Pyridyl KetoneThiosemicarbarzone, Metolazone, Minoxidil, Muzolimine, Pargyline,Pempidine, Pinacidil, Piperoxan, Primaperone, Protoveratrines,Raubasine, Rescimetol, Rilmenidene, Saralasin, Sodium Nitroprusside,Ticrynafen, Trimethaphan Camsylate, Tyrosinase, Urapidil and Valsartan;

Antihyperthyroids such as 2-Amino-4-methylthiazole, 2-Aminothiazole,Carbimazole, 3,5-Dibromo-L-tyrosine, 3,5-Diiodotyrosine, Hinderin,Iodine, lothiouracil, Methimazole, Methylthiouracil, Propylthiouracil,Sodium Perchlorate, Thibenzazoline, Thiobarbital and 2-Thiouracil;

Antihypotensive drugs such as Amezinium Methyl Sulfate, AngiotensinAmide, Dimetofrine, Dopamine, Etifelmin, Etilefrin, Gepefrine,Metaraminol, Midodrine, Norepinephrine, Pholedrinead and Synephrine;

Antihypothyroid drugs such as Levothyroxine Sodium, Liothyronine,Thyroid, Thyroidin, Thyroxine, Tiratricol and TSH;

Anti-Inflammatory (non-steroidal) drugs, including: Aminoarylcarboxylicacid derivatives such as Enfenamic Acid, Etofenamate, Flufenamic Acid,Isonixin, Meclofenamic Acid, Mefanamic Acid, Niflumic Acid,Talniflumate, Terofenamate and Tolfenamic Acid;

Arylacetic acid derivatives such as Acemetacin, Alclofenac, Amfenac,Bufexamac, Cinmetacin, Clopirac, Diclofenac Sodium, Etodolac, Felbinac,Fenclofenac, Fenclorac, Fenclozic Acid, Fentiazac, Glucametacin,Ibufenac, Indomethacin, Isofezolac, Isoxepac, Lonazolac, MetiazinicAcid, Oxametacine, Proglumetacin, Sulindac, Tiaramide, Tolmetin andZomepirac;

Arylbutyric acid derivatives such as Bumadizon, Butibufen, Fenbufen andXenbucin;

Arylcarboxylic acids such as Clidanac, Ketorolac and Tinoridine;

Arylpropionic acid derivatives such as Alminoprofen, Benoxaprofen,Bucloxic Acid, Carprofen, Fenoprofen, Flunoxaprofen, Flurbiprofen,Ibuprofen, Ibuproxam, Indoprofen, Ketoprofen, Loxoprofen, Miroprofen,Naproxen, Oxaprozin, Piketoprofen, Pirprofen, Pranoprofen, ProtizinicAcid, Suprofen and Tiaprofenic Acid;

Pyrazoles such as Difenamizole and Epirizole;

Pyrazolones such as Apazone, Benzpiperylon, Feprazone, Mofebutazone,Morazone, Oxyphenbutazone, Phenybutazone, Pipebuzone, Propyphenazone,Ramifenazone, Suxibuzone and Thiazolinobutazone;

Salicylic acid derivatives such as Acetaminosalol, Aspirin, Benorylate,Bromosaligenin, Calcium Acetylsalicylate, Diflunisal, Etersalate,Fendosal, Gentisic Acid, Glycol Salicylate, Imidazole Salicylate, LysineAcetylsalicylate, Mesalamine, Morpholine Salicylate, 1-NarhthylSalicylate, Olsalazine, Parsalmide, Phenyl Acetylsalicylate, PhenylSalicylate, Salacetamide, Salicylamine O-Acetic Acid, SalicylsulfuricAcid, Salsalate and Sulfasalazine;

Thiazinecarboxamides such as Droxicam, Isoxicam, Piroxicam andTenoxicam; and

others such as ∈-Acetamidocaproic Acid, S-Adenosylmethionine,3-Amino-4-hydroxybutyric Acid, Amixetrine, Bendazac, Benzydamine,Bucolome, Difenpiramide, Ditazol, Emorfazone, Guaiazulene, Nabumetone,Nimesulide, Orgotein, Oxaceprol, Paranyline, Perisoxal, Pifoxime,Proquazone, Proxazole and Tenidap;

Antimalarial drugs such as Acedapsone, Amodiaquin, Arteether,Artemether, Artemisinin, Artesunate, Bebeerine, Berberine, Chirata,Chlorguanide, Chloroquine, Chlorproguanil, Cinchona, Cinchonidine,Cinchonine, Cycloguanil, Gentiopicrin, Halofantrine, Hydroxychloroquine,Mefloquine Hydrochloride, 3-Methylarsacetin, Pamaquine, Plasmocid,Primaquine, Pyrimethamine, Quinacrine, Quinine, Quinine Bisulfate,Quinine Carbonate, Quinine Dihydrobromide, Quinine Dihydrochloride,Quinine Ethylcarbonate, Quinine Formate, Quinine Gluconate, QuinineHydriodide, Quinine Hydrochloride, Quinine Salicylate, Quinine Sulfate,Quinine Tannate, Quinine Urea Hydrochloride, Quinocide, Quinoline andSodium Arsenate Diabasic;

Antimigraine drugs such as Alpiropride, Dihydroergotamine, Eletriptan,Ergocornine, Ergocorninine, Ergocryptine, Ergot, Ergotamine,Flumedroxone acetate, Fonazine, Lisuride, Methysergid(e), Naratriptan,Oxetorone, Pizotyline, Rizatriptan and Sumatriptan;

Antinauseant drugs such as Acetylleucine Monoethanolamine, Alizapride,Benzquinamide, Bietanautine, Bromopride, Buclizine, Chlorpromazine,Clebopride, Cyclizine, Dimenhydrinate, Dipheniodol, Domperidone,Granisetron, Meclizine, Methalltal, Metoclopramide, Metopimazine,Nabilone, Ondansteron, Oxypendyl, Pipamazine, Piprinhydrinate,Prochlorperazine, Scopolamine, Tetrahydrocannabinols, Thiethylperazine,Thioproperzaine and Trimethobenzamide;

Antineoplastic drugs, including: Alkylating agents, such as Alkylsulfonates such as Busulfan, Improsulfan and Piposulfan;

Aziridines such as Benzodepa, Carboquone, Meturedepa and Uredepa;

Ethylenimines and methylmelamines such as Altretamine,Triethylenemelamine, Triethylenephosphoramide,Triethylenethiophosphoramide and Trimethylolomelamine;

Nitrogen mustards such as Chlorambucil, Chlornaphazine,Chclophosphamide, Estramustine, Ifosfamide, Mechlorethamine,Mechlorethamine Oxide Hydrochloride, Melphalan, Novembichin,Phenesterine, Prednimustine, Trofosfamide and Uracil Mustard;

Nitrosoureas such as Carmustine, Chlorozotocin, Fotemustine, Lomustine,Nimustine and Ranimustine; and

others such as Camptothecin, Dacarbazine, Mannomustine, Mitobronitol,Mitolactol and Pipobroman;

Antibiotics such as Aclacinomycins, Actinomycin F₁, Anthramycin,Azaserine, Bleomycins, Cactinomycin, Carubicin, Carzinophilin,Chromomycins, Dactinomycin, Daunorubicin, 6-Diazo-5-oxo-L-norleucine,Doxorubicin, Epirubicin, Mitomycins, Mycophenolic Acid, Nogalamycin,Olivomycins, Peplomycin, Plicamycin, Porfiromycin, Puromycin,Streptonigrin, Streptozocin, Tubercidin, Ubenimex, Zinostatin andZorubicin;

Antimetabolites, including: Folic acid analogs such as Denopterin,Methotrexate, Pteropterin and Trimetrexate;

Purine analogs such as Fludarabine, 6-Mercaptopurine, Thiamiprine andThioguanaine; and

Pyrimidine analogs such as Ancitabine, Azacitidine, 6-Azauridine,Carmofur, Cytarabine, Doxifluridine, Enocitabine, FloxuridineFluroouracil and Tegafur;

Enzymes such as L-Asparaginase; and

others such as Aceglatone, Amsacrine, Bestrabucil, Bisantrene,Bryostatin 1, Carboplatin, Cisplatin, Defofamide, Demecolcine,Diaziquone, Elfornithine, Elliptinium Acetate, Etoglucid, Etoposide,Gallium Nitrate, Hydroxyurea, Interferon-α, Interferon-β, Interferon-γ,Interleukine-2, Lentinan, Letrozole, Lonidamine, Mitoguazone,Mitoxantrone, Mopidamol, Nitracrine, Pentostatin, Phenamet, Pirarubicin,Podophyllinicc Acid, 2-Ethythydrazide, Polynitrocubanes, Procarbazine,PSK7, Razoxane, Sizofiran, Spirogermanium, Taxol, Teniposide, TenuazonicAcid, Triaziquone, 2.2′.2″-Trichlorotriethylamine, Urethan, Vinblastine,Vincristine, Vindesine and Vinorelbine;

Antineoplastic (hormonal) drugs, including: Androgens such asCalusterone, Dromostanolone Propionate, Epitiostanol, Mepitiostane andTestolactone;

Antiadrenals such as Aminoglutethimide, Mitotane and Trilostane;

Antiandrogens such as Flutamide and Nilutamide; and

Antiestrogens such as Tamoxifen and Toremifene;

Antineoplastic adjuncts including folic acid replenishers such asFrolinic Acid;

Antiparkinsonian drugs such as Amantadine, Benserazide, Bietanautine,Biperiden, Bromocriptine, Budipine, Cabergoline, Carbidopa, Deprenyl(a/k/a L-deprenyl, L-deprenil, L-deprenaline and selegiline),Dexetimide, Diethazine, Diphenhydramine, Droxidopa, Ethopropazine,Ethylbenzhydramine, Levodopa, Naxagolide, Pergolide, Piroheptine,Pramipexole, Pridinol, Prodipine, Quinpirole, Remacemide, Ropinirole,Terguride, Tigloidine and Trihexyphenidyl Hydrochloride;

Antipheochromocytoma drugs such as Metyrosine, Phenoxybenzamine andPhentolamine;

Antipneumocystis drugs such as Effornithine, Pentamidine andSulfamethoxazole;

Antiprostatic hypertrophy drugs such as Gestonorone Caproate,Mepartricin, Oxendolone and Proscar7;

Antiprotozoal drugs (Leshmania) such as Antimony Sodium Gluconate,Ethylstibamine, Hydroxystilbamidine, N-Methylglucamine, Pentamidine,Stilbamidine and Urea Stibamine;

Antiprotozoal drugs (Trichomonas) such as Acetarsone, Aminitrozole,Anisomycin, Azanidazole, Forminitrazole, Furazolidone, Hachimycin,Lauroguadine, Mepartricin, Metronidazole, Nifuratel, Nifuroxime,Nimorazole, Secnidazole, Silver Picrate, Tenonitrozole and Tinidazole;

Antiprotozoal drugs (Trypanosma) such as Benznidazole, Eflornithine,Melarsoprol, Nifurtimox, Oxophenarsine, Hydrochloride, Pentamidine,Propamidine, Puromycin, Quinapyramine, Stilbamidine, Suramin Sodium,Trypan Red and Tryparasmide;

Antipuritics such as Camphor, Cyproheptadine, Dichlorisone, Glycine,Halometasone, 3-Hydroxycamphor, Menthol, Mesulphen, Methdilazine,Phenol, Polidocanol, Risocaine, Spirit of Camphor, Thenaldine,Tolpropamine and Trimeprazine;

Antipsoriatic drugs such as Acitretin, Ammonium Salicylate, Anthralin,6-Azauridine, Bergapten(e), Chrysarobin, Etretinate and Pyrogallol;

Antipsychotic drugs, including: Butyrophenones such as Benperidol,Bromperidol, Droperidol, Fluanisone, Haloperidol, Melperone, Moperone,Pipamperone, Sniperone, Timiperone and Trifluperidol;

Phenothiazines such as Acetophenazine, Butaperazine, Carphenazine,Chlorproethazine, Chlorpromazine, Clospirazine, Cyamemazine, Dixyrazine,Fluphenazine, Imiclopazine, Mepazine, Mesoridazine, Methoxypromazine,Metofenazate, Oxaflumazine, Perazine, Pericyazine, Perimethazine,Perphenazine, Piperacetazine, Pipotiazine, Prochlorperazine, Promazine,Sulforidazine, Thiopropazate, Thioridazine, Trifluoperazine andTriflupromazine;

Thioxanthenes such as Chlorprothixene, Clopenthixol, Flupentixol andThiothixene;

other tricyclics such as Benzquinamide, Carpipramine, Clocapramine,Clomacran, Clothiapine, Clozapine, Opipramol, Prothipendyl,Tetrabenazine, and Zotepine; and

others such as Alizapride, Amisulpride, Buramate, Fluspirilene,Molindone, Penfluridol, Pimozide, Spirilene and Sulpiride;

Antipyretics such as Acetaminophen, Acetaminosalol, Acetanilide,Aconine, Aconite, Aconitine, Alclofenac, Aluminum Bis(acetylsalicylate),Aminochlorthenoxazin, Aminopyrine, Aspirin, Benorylate, Benzydamine,Berberine, p-Bromoacetanilide, Bufexamac, Bumadizon, CalciumAcetysalicylate, Chlorthenoxazin(e), Choline Salicylate, Clidanac,Dihydroxyaluminum Acetylsalicylate, Dipyrocetyl, Dipyrone, Epirizole,Etersalate, Imidazole Salicylate, Indomethacin, Isofezolac,p-Lactophenetide, Lysine Acetylsalicylate, Magnesium Acetylsalicylate,Meclofenamic Acid, Morazone, Morpholine Salicylate, Naproxen,Nifenazone, 51-Nitro-2′-propoxyacetanilide, Phenacetin, Phenicarbazide,Phenocoll, Phenopyrazone, Phenyl Acetylsalicylate, Phenyl Salicylate,Pipebuzone, Propacetamol, Propyphenazone, Ramifenazone, Salacetamide,Salicylamide O-Acetic Acid, Sodium Salicylate, Sulfamipyrine,Tetrandrine and Tinoridine;

Antirickettsial drugs such as p-Aminobenzoic Acid, Chloramphenicol,Chloramphenicol Palmitate, Chloramphenicol Pantothenate andTetracycline;

Antiseborrheic drugs such as Chloroxine, 3-O-Lauroylpyridoxol Diacetate,Piroctone, Pyrithione, Resorcinol, Selenium Sulfides and Tioxolone;

Antiseptics, including: Guanidines such as Alexidine, Ambazone,Chlorhexidine and Picloxydine;

Halogens and halogen compounds such as Bismuth Iodide Oxide, Bismuthlodosubgallate, Bismuth Tribromophenate, Bornyl Chloride, CalciumIodate, Chlorinated Lime, Cloflucarban, Flurosalan, Iodic Acid, Iodine,Iodine Monochloride, Iodine Trichloride, Iodoform, MethenamineTetraiodine, Oxychlorosene, Povidone-Iodine, Sodium Hypochlorite, SodiumIodate, Symclosene, Thymol Iodide, Triclocarban, Triclosan andTroclosene Potassium;

Mercurial compounds such as Hydragaphen, Meralein Sodium, Merbromin,Mercuric Chloride, Mercuric Chloride, Ammoniated, Mercuric Sodiump-Phenolsulfonate, Mercuric Succinimide, Mercuric Sulfide, Red,Mercurophen, Mercurous Acetate, Mercurous Chloride, Mercurous Iodide,Nitromersol, Potassium Tetraiodomercurate(II), PotassiumTriiodomercurate (II) Solution, Thimerfonate Sodium and Thimerosal;

Nitrofurans such as Furazolidone, 2-(Methoxymethyl)-5-nitrofuran,Nidroxyzone, Nifuroxime, Nifurzide and Nitrofurazone;

Phenols such as Acetomeroctol, Bithionol, Cadmium Salicylate, Carvacrol,Chloroxylenol, Clorophene, Cresote, Cresol(s), p-Cresol, Fenticlor,Hexachlorophene, 1-Napthyl Salicylate, 2-Napthyl Salicylate,2,4,6-Tribromo-m-cresol, and 3′,4′,5′-Trichlorosalicylanilide;

Quinolines such as Aminoquinuride, Benzoxiquine, Broxyquinoline,Chloroxine, Chlorquinaldol, Cloxyquin, Ethylhydrocupreine, Euprocin,Halquinol, Hydrastine, 8-Hydroxquinoline, 8-Hydroxquinoline Sulfate andIodochlorhydroxyquin; and

others such as Aluminum Acetate Solution, Aluminum Subacetate Solution,Aluminum Sulfate, 3-Amino-4-hydroxybutyric Acid, Boric Acid,Chlorhexidine, Chloroazodin, m-Cresyl Acetate, Cupric Sulfate,Dibromopropamidine, Ichthammol, Negatol7, Noxytiolin, Ornidazole,β-Propiolactone, α-Terpineol;

Antispasmodic drugs such as Alibendol, Ambucetamide,

Aminopromazine, Apoatropine, Bevonium Methyl Sulfate, Bietamiverine,Butaverine, Butropium Bromide, N-Butylscopolammonium Bromide,Caroverine, Cimetropium Bromide, Cinnamedrine, Clebopride, ConiineHydrobromide, Coniine Hydrochloride, Cyclonium Iodide, Difemerine,Diisopromine, Dioxaphetyl Butyrate, Diponium Bromide, Drofenine,Emepronium Bromide, Ethaverine, Feclemine, Fenalamide, Fenoverine,Fenpiprane, Fenpiverinium Brcmide, Fentonium Bromide, Flavoxate,Flopropione, Gluconic Acid, Guaiactamine, Hydramitrazine, Hymecromone,Leiopyrrole, Mebeverine, Moxaverine, Nafiverine, Octamylamine,Octaverine, Pentapiperide, Phenamacide Hydrochloride, Phloroglucinol,Pinaverium Bromide, Piperilate, Pipoxolan Hydrochloride, Pramiverin,Prifinium Bromide, Properidine, Propivane, Propyromazine, Prozapine,Racefemine, Rociverine, Spasmolytol, Stilonium Iodide, Sultroponium,Tiemonium Iodide, Tiquizium Bromide, Tiropramide, Trepibutone,Tricromyl, Trifolium, Trimebutine,N,N-ITrimethyl-3,3-diphenyl-propylamine, Tropenzile, Trospium Chlorideand Xenytropium Bromide;

Antithrombotic drugs such as Anagrelide, Argatroban, Cilostazol,Chrysoptin, Daltroban, Defibrotide, Enoxaparin, Fraxiparine7, Indobufen,Lamoparan, Ozagrel, Picotamide, Plafibride, Reviparin, Tedelparin,Ticlopidine, Triflusal and Warfarin;

Antitussive drugs such as Allocamide, Amicibone, Benproperine,Benzonatate, Bibenzonium Bromide, Bromoform, Butamirate, Butethamate,Caramiphen Ethanedisulfonate, Carbetapentane, Chlophedianol, Clobutinol,Cloperastine, Codeine, Codeine Methyl Bromide, Codeine N-Oxide, CodeinePhosphate, Codeine Sulfate, Cyclexanone, Dextromethorphan, DibunateSodium, Dihydrocodeine, Dihydrocodeinone Enol Acetate, Dimemorfan,Dimethoxanate, α,α-Diphenyl-2-piperidinepropanol, Dropropizine,Drotebanol, Eprazinone, Ethyl Dibunate, Ethylmorphine, Fominoben,Guiaiapate, Hydrocodone, Isoaminile, Levopropoxyphene, Morclofone,Narceine, Normethadone, Noscapine, Oxeladin, Oxolamine, Pholcodine,Picoperine, Pipazethate, Piperidione, Prenoxdiazine Hydrochloride,Racemethorphan, Taziprinone Hydrochloride, Tipepidine and Zipeprol;

Antiulcerative drugs such as Aceglutamide Aluminum Complex,∈-Acetamidocaproic Acid Zinc Salt, Acetoxolone, Arbaprostil, BenexateHydrochloride, Bismuth Subcitrate Sol (Dried), Carbenoxolone, Cetraxate,Cimetidine, Enprostil, Esaprazole, Famotidine, Ftaxilide, Gefarnate,Guaiazulene, Irsogladine, Misoprostol, Nizatidine, Omeprazole,Ornoprostil, γ-Oryzanol, Pifarnine, Pirenzepine, Plaunotol, Ranitidine,Rioprostil, Rosaprostol, Rotraxate, Roxatidine Acetate, Sofalcone,Spizofurone, Sucralfate, Teprenone, Trimoprostil, Thrithiozine,Troxipide and Zolimidine;

Antiurolithic drugs such as Acetohydroxamic Acid, Allopurinol, PotassiumCitrate and Succinimide;

Antivenin drugs such as Lyovac7 Antivenin;

Antiviral drugs, including: Purines and pyrimidinones such as Acyclovir,Cytarabine, Dideoxyadenosine, Dideoxycytidine, Dideoxyinosine,Edoxudine, Floxuridine, Ganciclovir, Idoxuridine, Inosine Pranobex,MADU, Penciclovir, Trifluridine, Vidrarbine and Zidovudiine; and

others such as Acetylleucine Monoethanolamine, Amantadine, Amidinomycin,Cosalane, Cuminaldehyde Thiosemicarbzone, Foscarnet Sodium, Imiquimod,Interferon-α, Interferon-β, Interferon-γ, Kethoxal, Lysozyme,Methisazone, Moroxydine, Podophyllotoxin, Ribavirin, Rimantadine,Stallimycin, Statolon, Tromantadine and Xenazoic Acid;

Anxiolytic drugs, including: Arylpiperazines such as Buspirone,Gepirone, Ipsapirone and Tondospirone;

Benzodiazepine derivatives such as Alprazolam, Bromazepam, Camazepam,Chlordiazepoxide, Clobazam, Clorazepate, Chotiazepam, Cloxazolam,Diazepam, Ethyl Loflazepate, Etizolam, Fluidazepam, Flutazolam,Flutoprazepam, Halazepam, Ketazolam, Lorazepam, Loxapine, Medazepam,Metaclazepam, Mexazolam, Nordazepam, Oxazepam, Oxazolam, Pinazepam,Prazepam and Tofisopam;

Carbamates such as Cyclarbamate, Emylcamate, Hydroxyphenamate,Meprobamate, Phenprobamate and Tybamate; and

others such as Alpidem, Benzoctamine, Captodiamine, Chlormezanone,Etifoxine, Flesinoxan, Fluoresone, Glutamic Acid, Hydroxyzine,Lesopitron, Mecloralurea, Mephenoxalone, Mirtazepine, Oxanamide,Phenaglycodol, Suriclone and Zatosetron;

Benzodiazepine antagonists such as Flumazenil;

Bronchodilators, including: Ephedrine derivatives such as Albuterol,Bambuterol, Bitolterol, Carbuterol, Clenbuterol, Clorprenaline,Dioxethedrine, Ephedrine, Epiniphrine, Eprozinol, Etafedrine,Ethylnorepinephrine, Fenoterol, Hexoprenaline, Isoetharine,Isoproterenol, Mabuterol, Metaproterenol, N-Methylephedrine, Pirbuterol,Procaterol, Protokylol, Reproterol, Rimiterol, Salmeterol, Soterenol,Terbutaline and Tulobuterol;

Quaternary ammonium compounds such as Bevonium Methyl Sulfate,Clutropium Bromide, Ipratropium Bromide and Oxitropium Bromide;

Xanthine derivatives such as Acefylline, Acefylline Piperazine,Ambuphylline, Aminophylline, Bamifylline, choline Theophyllinate,Doxofylline, Dyphylline, Enprofylline, Etamiphyllin, Etofylline,Guaithylline, Proxyphylline, Theobromine, 1-Theobromineacetic Acid andTheophylline; and

others such as Fenspiride, Medibazine, Montekulast, Methoxyphenanime,Tretoquinol and Zafirkulast;

Calcium channel blockers, including: Arylalkylamines such as Bepridil,Ditiazem, Fendiline, Gallopanil, Prenylamine, Terodiline and Verapamil;

Dihydropyridine derivatives such as Felodipine, Isradipine, Nicardipine,Nifedipine, Nilvadipine, Nimodipine, Nisoldipine and Nitrendipine;

Piperazine derivatives such as Cinnarizine, Flunarisine and Lidoflazine;and

others such as Bencyclane, Etafenone and Perhexiline;

Calcium regulators such as Calcifediol, Calcitonin, Calcitriol,Clodronic Acid, Dihydrotachysterol, Elcatonin, Etidronic Acid,Ipriflavone, Pamidronic Acid, Parathyroid Hormone and TeriparatideAcetate;

Cardiotonics such as Acefylline, Acetyldigititoxins, 2-Amino-4-picoline,Amrinone, Benfurodil Hemisuccinate, Buclasdesine, Cerberoside,Camphotamide, Convallatoxin, Cymarin, Denopamine, Deslanoside, Ditalin,Digitalis, Digitoxin, Digoxin, Dobutamine, Dopamine, Dopexamine,Enoximone, Erythrophleine, Fenalcomine, Gitalin, Gitoxin, Glycocyamine,Heptaminol, Hydrastinine, Ibopamine, Lanotodises, Metamivam, Milrinone,Neriifolin, Oleandrin, Ouabain, Oxyfedrine, Prenalterol, Proscillaridin,Resibufogenin, Scillaren, Scillarenin, Strophanthin, Sulmazole,Theobromine and Xamoterol;

Chelating agents such as Deferozmine, Ditiocarb Sodium, Edetate CalciumDisodium, Edetate Disodium, Edeate Sodium, Edetate Trisodium,Penicillamine, Pentetate Calcium Trisodium, Pentectic Acid, Succimer andTrientine;

Cholecystokinin antagonists such as Proglumide;

Cholelitholytic agents such as Chenodiol, Methyl tert-Butyl Ether,Monooctanoin and Ursodiol;

Choleretics such as Alibendol, Anethole Trithion, Azintamide, CholicAcid, Cicrotoic Acid, Clanobutin, Cyclobutyrol, Cyclovalone, Cynarin(e),Dehydrocholic Acid, Deoxycholic Acid, Dimecrotic Acid, α-EthylbenzylAlcohol, Exiproben, Feguprol, Fencibutirol, Fenipentol, Florantyrone,Hymecromone, Menbutone, 3-(o-Methoxyphenyl)-2-phenylacrylic Acid,Metochalcone, Moquizone, Osalmid, Ox Bile Extract, 4.4′-Oxydi-2-butanol,Piprozolin, Prozapine, 4-Salicyloylmorpholine, Sincalide, TaurocholicAcid, Timonacic, Tocamphyl, Trepibutone and Vanitiolide;

Cholinergic agents such as Aceclidine, Acetylcholine Bromide,Acetylcholide Chloride, Aclatonium Napadisilate, Benzpyrinium Bromide,Bethanechol chloride, Carbachol, Carpronium chloride, DemecariumBromide, Dexpanthenol, Diisopropyl Paraoxon, Echothiophate Iodide,Edrophomium chloride, Eseridine, Furtrethonium, Isoflurophate,Methacholine chloride, Muscarine, Neostigmine, Oxapropanium Iodide,Physostigmine and Pyridostigmine Bromide;

Cholinesterase inhibitors such as Ambenonium Chloride, DistigmineBromide and Galanthamine;

Cholinesterase reactivators such as Obidoximine Chloride and PralidoximeChloride;

Central nervous system stimulants and agents such as Amineptine,Amphetimine, Amphetaminil, Bemegride, Benzphetamine, Brucine, Caffeine,Chlorphentermine, Clofenciclan, Clortermine, Coca, Demanyl Phosphate,Dexoxadrol, Dextroamphetamine Sulfate, Diethlpropion,N-Ethylamphetamine, Ethamivan, Etifelmin, Etryptamine, Fencamfamine,Fenethylline, Fenosolone, Flurothyl, Galanthamine, Hexacyclonate Sodium,Homocamfin, Mazindol, Megexamide, Methamphetamine, Methylphenidate,Nikethamide, Pemoline, Pentylenetetrazole, Phenidimetrazine,Phenmetrazine, Phentermine, Picrotoxin, Pipradrol, Prolintane andPyrovalerone;

Decongestants such as Amidephrine, Cafaminol, Cyclopentamine, Ephedrine,Epinephrine, Fenoxazoline, Indanazoline, Metizoline, Naphazoline,Nordefrin Hydrochloride, Octodrine, oxymetazoline, PhenylephrineHydrochloride, Phenylpropanolamine Hydrochloride,Phenylpropylmethylamine, Propylhexedrine, Pseudoephedrine,Tetrahydrozoline, Tymazoline and Xylometazoline;

Dental agents, including: Bisphosphonates (anti-periodontal disease andbone resorption) such as Alendronate, Clodronate, Etidronate,Pamidronate and Tiludronate; Carries Prophylactics such as Arginine andSodium Fluoride;

Desensitizing Agents such as Potassium Nitrate and Citrate Oxalate;

Depigmentors such as Hydroquinine, Hydroquinone and Monobenzone;

Diuretics, including: Organomercurials such as Chlormerodrin,Meralluride, Mercamphamide, Mercaptomerin Sodium, Mercumallylic Acid,Mercumatilin Sodium, Mercurous Chloride and Mersalyl;

Pteridines such as Furterene and Triamterene;

Purines such as Acefylline, 7-Morpholinomethyltheophylline, Pamabrom,Protheobromine and Theobromine;

Steroids such as Canrenone, Oleandrin and Spironolactone;

Sulfonamide derivatives such as Acetazolmide, Ambuside, Azosemide,Bumetanide, Butazolamide, Chloraminophenamide, Clofenamide, Clopamide,Clorexolene, Diphenylmethane-4.4′-disulfonamide, Disulfamide,Ethbxzolamide, Furosemide, Indapamide, Mefruside, Methazolamide,Piretanide, Quinethazone, Torasemide, Tripamide and Xipamide;

Uracils such as Aminometradine and Amisometradine;

others such as Amanozine, Amiloride, Arbutin, Chlorazanil, EthacrynicAcid, Etozolin, Hydracarbazine, Isosorbide, Mannitol, Metochalcone,Muzolimine, Perhexiline, Ticrynafen and Urea;

Dopamine receptor agonists such as Bromocriptine, Dopexamine,Fenoldopam, Ibopamine, Lisuride, Naxagolide and Pergolide;

Ectoparasiticides such as Amitraz, Benzyl Benzoate, Carbaryl,Crotamiton, DDT, Dixanthogen, Isobornyl Thiocyanoacetate—Technical, LimeSulfurated Solution, Lindane, Malathion, Mercuric Oleate, Mesulphen andSulphur—Pharmaceutical;

Enzymes, including: Digestive enzymes such as α-Amylase (SwinePancreas), Lipase, Pancrelipase, Pepsin and Rennin;

Mucolytic enzymes such as Lysozyme;

Penicillin inactivating enzymes such as Penicillinase; and

Proteolytic enzymes such as Collagenase, Chymopapain, Chymotrypsins,Papain and Trypsin;

Enzyme inducers (hepatic) such as Flumecinol;

Estrogens, including: Nonsteroidal estrogens such as Benzestrol,Broparoestrol, Chlorotrianisene, Dienestrol, Diethylstilbestrol,Diethylstilbestrol Diproprionate, Dimestrol, Fosfestrol, Hexestrol,Methallenestril and Methestrol; and

Steroidal estrogens such as Colpormon, Conjugated Estrogenic Hormones,Equilenin, Equilin, Estradiol, Estradiol Benzoate, Estradiol17β-Cypionate, Estriol, Estrone, Ethinyl Estradiol, Mestranol,Moxestrol, Mytatrienediol, Quinestradiol and Quinestrol;

Gastric secretion inhibitors such as Enterogastrone and Octreotide;

Glucocorticoids such as 21-Acetoxyprefnenolone, Aalclometasone,Algestone, Amicinonide, Beclomethasone, Betamethasone, Budesonide,Chloroprednisone, Clobetasol, Blovetasone, Clocortolone, Cloprednol,Corticosterone, Cortisone, Cortivazol, Deflazacort, Desonide,Desoximetasone, Dexamethasone, Diflorasone, Diflucortolone,Difluprednate, Enoxolone, Fluazacort, Flucloronide, Flumehtasone,Flunisolide, Fluocinolone Acetonide, Fluocinonide, Fluocortin Butyl,Fluocortolone, Fluorometholone, Fluperolone Acetate, FluprednideneAcetate, Fluprednisolone, Flurandrenolide, Formocortal, Halcinonide,Halometasone, Halopredone Acetate, Hydrocortamate, Hydrocortisone,Hydrocortisone Acetate, ydrocortisone Phosphate, Hydrocortisone21-Sodium Succinate, Hydrocortisone Tebutate, Mazipredone, Medrysone,Meprednisone, Methyolprednisolone, Mometasone Furoate, Paramethasone,Prednicarbate, Prednisolone, Prednisolone 21-Diethylaminoacetate,Prednisone Sodium Phosphate, Prednisolone Sodium Succinate, PrednisoloneSodium 21-m-Sulfobenzoate, Prednisolone 21-Stearoylglycolate,Prednisolone Tebutate, Prednisolone 21-Trimethylacetate, Prednisone,Prednival, Prednylidene, Prednylidene 21-Diethylaminoacetate,Tixocortal, Triamcinolone, Triamcinolone Acetonide, TriamcinoloneBenetonide and Triamcinolone Hexacetonide;

Gonad-Stimulating principles such as Buserelin, Clomiphene, Cyclofenil,Epimestrol, FSH, HCG and LH-RH;

Gonadotropic hormones such as LH and PMSG;

Growth hormone inhibitors such as Octreotide and Somatostatin;

Growth hormone releasing factors such as Semorelin;

Growth stimulants such as Somatotropin;

Hemolytic agents such as Phenylhydrazine and PhenylhydrazineHydrochloride;

Heparin antagonists such as Hexadimethrine Bromide and Protamines;

Hepatoprotectants such as S-Adenosylmethionine, Betaine, Catechin,Citolone, Malotilate, Orazamide, Phosphorylcholine, Protoporphyrin IX,Silymarin-Group, Thiotic Acid and Tiopronin;

Immunomodulators such as Amiprilose, Bucillamine, Ditiocarb Sodium,Inosine Pranobex, Interferon-γ, Interleukin-2, Lentinan, Muroctasin,Platonin, Procodazole, Tetramisole, Thymomodulin, Thymopentin andUbenimex;

Immunosuppressants such as Azathioprine, Cyclosporins and Mizoribine;

Ion exchange resins such as Carbacrylic Resins, Cholestyramine Resin,Colestipol, Polidexide, Resodec and Sodium Polystyrene Sulfonate;

Lactation stimulating hormone such as Prolactin;

LH-RH agonists such as Buserelin, Goserelin, Leuprolide, Nafarelin, andTriptorelin;

Lipotropic agents such as N-Acetylmethionine, Choline Chloride, CholineDehydrocholate, Choline Dihydrogen Citrate, Inositol, Lecithin andMethionine;

Lupus erythematosus suppressants such as Bismuth Sodium Triglycollamate,Bismuth Subsalicylate, Chloroquine and Hydroxychloroquine;

Mineralcorticoids such as Aldosterone, Deoxycorticosterone,Deoxycorticosterone Acetate and Fludrocortisone;

Miotic drugs such as Carbachol, Physostigmine, Pilocarpine andPilocarpus;

Monoamine oxidase inhibitors such as Deprenyl, Iproclozide, Iproniazid,Isocarboxazid, Moclobemide, Octomoxin, Pargyline, Phenelzine,Phenoxypropazine, Pivalylbenzhydrazine, Prodipine, Toloxatone andTranylcypromine;

Mucolytic agents such as Acetylcysteine, Bromhexine, Carbocysteine,Domiodol, Letosteine, Lysozyme, Mecysteine Hydrochloride, Mesna,Sobrerol, Stepronin, Tiopronin and Tyloxapol;

Muscle relaxants (skeletal) such as Afloqualone, Alcuronium, AtracuriumBesylate, Baclofen, Benzoctamine, Benzoquinonium Chloride,C-Calebassine, Carisoprodol, Chlormezanone, Chlorphenesin Carbamate,Chlorproethazine, Chlozoxazone, Curare, Cyclarbamate, Cyclobenzaprine,Dantrolene, Decamethonium Bromide, Diazepam, Eperisone, FazadiniumBromide, Flumetramide, Gallamine Triethiodide, Hexacarbacholine Bromide,Hexafluorenium Bromide, Idrocilamide, Lauexium Methyl Sulfate,Leptodactyline, Memantine, Mephenesin, Mephenoxalone, Metaxalone,Methocarbamol, Metocurine Iodide, Nimetazepam, Orphenadrine, PancuroniumBromide, Phenprobamate, Phenyramidol, Pipecurium Bromide, Promoxolane,Quinine Sulfate, Styramate, Succinylcholine Bromide, SuccinylcholineChloride, Succinylcholine Iodine, Suxethonium Bromide, Tetrazepam,Thiocolchicoside, Tizanidine, Tolperisone, Tubocurarine Chloride,Vecuronium Bromide and Zoxolamine;

Narcotic antagonists such as Amiphenazole, Cyclazocine, Levallorphan,Nadide, Nalmfene, Nalorphine, Nalorphine Dinicotinate, Naloxone andNaltrexone;

Neuroprotective agents such as Dizocilpine;

Nootropic agents such as Aceglutamide, Acetylcarnitine, Aniracetam,Bifematlane, Exifone, Fipexide, Idebenone, Indeloxazune Hydrochloride,Nizofenone, Oxiracetam, Piracetam, Propentofylline, Pyritinol andTacrine;

Ophthalmic agents such as 15-ketoprostaglandins;

Ovarian hormone such as Relaxin;

Oxytocic drugs such as Carboprost, Cargutocin, Deaminooxytocin,Ergonovine, Gemeprost, Methylergonovine, Oxytocin, Pituitary(Posterior), Prostaglandin E₂, Prostaglandin F_(2a) and Sparteine;

Pepsin inhibitors such as Sodium Amylosulfate;

Peristaltic stimulants such as Cisapride;

Progestogens such as Allylestrenol, Anagestone, Chlormadinone Acetate,Delmadinone Acetate, Demegestone, Desogestrel, Dimethisterone,Dydrogesterone, Ethisterone, Ethynodiol, Flurogestone Acetate,Gestodene, Gestonorone Caproate, Haloprogesterone,17-Hydroxy-16-methylene—progesterone, 17α-Hydroxyprogesterone,17α-Hydroxygesterone Caproate, Lynestrenol, Medrogestone,Medroxyprogesterone, Megestrol Acetate, Melengestrol, Norethindrone,Norethynodrel, Norgesterone, Norgestimate, Norgestrel, Norgestrienone,Norvinisterone, Pentagestrone, Progesterone, Promegestone, Quingestroneand Trengestone;

Prolactin inhibitors such as Metergoline;

Prostaglandins and prostaglandin analogs such as Arbaprostil,Carboprost, Enprostil, Bemeprost, Limaprost, Misoprostol, Ornoprostil,Prostacyclin, Prostaglandin E1, Prostaglandin E2, Prostagland in Fla,Rioprostil, Rosaprostol, Sulprostone and Trimoprostil;

Protease inhibitors such as Aprotinin, Camostat, Gabexate andNafamostat;

Respiratory stimulants such as Almitrine, Bemegride, Carbon Dioxide,Cropropamide, Crotethamide, Dimefline, Dimorpholamine, Doxapram,Ethamivan, Fominoben, Lobeline, Mepixanox, Metamivam, Nikethamide,Picrotoxin, Pimeclone, Pyridofylline, Sodium Succinate and Tacrine;

Sclerosing agents such as Ethanolamine, Ethylamine, 2-HexyldecanoicAcid, Polidocanol, Quinine Bisulfate, Quinine Urea Hydrochloride, SodiumRicinoleate, Sodium Tetradecyl Sulfate and Tribenoside;

Sedatives and hypnotics, including: Acyclic ureides such asAcecarbromal, Apronalide, Bomisovalum, Capuride, Carbromal andEctylurea;

Alcohols such as Chlorhexadol, Ethchlorvynol, Meparfynol,4-Methyl-5-thiazoleethanol, tert-Pentyl Alcohol and2,2,2-Trichloroethanol;

Amides such as Butoctamide, Diethylbromoacetamide, Ibrotamide,Isovaleryl Diethylamide, Niaprazine, Tricetamide, Trimetozine, Zolpidemand Zopiclone;

Barbituric acid derivatives such as Allobarbital, Amobarbital,Aprobarbital, Barbital, Brallabarbital, Butabarbital Sodium, Butalbital,Butallylonal, Butethal, Carbubarb, Cyclobarbital, Cyclopentobarbital,Enallylpropymal, 5-Ethyl-5-(1-piperidyl) barbituric Acid,5-Furfuryl-5-isopropylbarbituric Acid, Heptabarbital, Hexethal Sodium,Hexobarbital, Mephobarbital, Methitural, Narcobarbital, Nealbarbital,Pentobarbital Sodium, Phenallymal, Phenobarbital, Phenobarbital Sodium,Phenylmethylbarbituric Acid, Probarbital, Propallylonal, Proxibarbal,Reposal, Secobarbital Sodium, Talbutal, Tetrabarbital, VinbarbitalSodium and Vinylbital;

Benzodiazepine derivatives such as Brotizolam, Doxefazepam, Estazolam,Flunitrazepam, Flurazepam, Haloxazolam, Loprazolam, Lormetazepam,Nitrazepam, Quazepam, Temazepam and Triazolam;

Bromides such as Ammonium Bromide, Calcium Bromide, CalciumBromolactobionate, Lithium Bromide, Magnesium Bromide, Potassium Bromideand Sodium Bromide;

Carbamates such as Amyl Carbamate—Tertiary, Ethinamate, Hexaprpymate,Meparfynol Carbamate, Novonal and Tricholorourethan;

Chloral derivatives such as Carbocloral, Chloral Betaine, ChloralFormamide, Chloral Hydrate, Chloralantipyrine, Dichloralphenazone,Pentaerythritol Chloral and Triclofos;

Piperidinediones such as Glutehimide, Methyprylon, Piperidione,Pyrithyldione, Taglutimide and Thalidomide;

Quinazolone derivatives such as Etaqualone, Mecloqualone andMethaqualone; and

others such as Acetal, Acetophenone, Aldol, Ammonium Valerate,Amphenidone, d-Bornyl α-Bromoisovalerate, d-Bornyl Isovalerate,Bromoform, Calcium 2-Ethylbutanoate, Carfinate, α-Chlorolose,Clomethiazole, Cypripedium, Doxylamine, Etodroxizine, Etomidate,Fenadiazole, Homofenazine, Hydrobromic Acid, Mecloxamine, MenthylValerate, Opium, Paraldehyde, Perlapine, Propiomazine, Rilmazafone,Sodium Oxybate, Sulfonethylmethane and Sulfonmethane;

Thrombolytic agents such as APSAC, Plasmin, Pro-Urokinase,Streptokinase, Tissue Plasminogen Activator and Urokinase;

Thyrotropic hormones such as TRH and TSH;

Uricosurics such as Benzbromarone, Ethebenecid, Orotic Acid,Oxycinchophen, Probenecid, Sulfinpyrazone, Ticrynafen and Zoxazolamine;

Vasodilators (cerebral) such as Bencyclane, Cinnarizine, Citicoline,Cyclandelate, Ciclonicate, Diisopropylamine Dichloractetate,Eburnamorine, Fenoxedil, Flunarizine, Ibudilast, Ifenprodil, Nafronyl,Nicametate, Nicergoline, Nimodipine, Papaverine, Pentifylline,Tinofedrine, Vincamine, Vinpocetine and Viquidil;

Vasodilators (coronary) such as Amotriphene, Bendazol, BenfurodilHemisuccinate, Benziodarone, Chloacizine, Chromonar, Clobenfurol,Clonitrate, Dilazep, Dipyridamole, Droprenilamine, Efloxate, Erythritol,Erythrityl Tetranitrate, Etafenone, Fendiline, Floredil, Ganglefene,Hexestrol Bis(β-diethylaminoethyl ether), Hexobendine, Itramin Tosylate,Khellin, Lidoflazine, Mannitol Hexanitrate, Medibazine, Nicorandil,Nitroglycerin, Pentaerythritol Tetranitrate, Pentrinitrol, Perhexiline,Pimefylline, Prenylamine, Propatyl Nitrate, Pyridofylline, Trapidil,Tricromyl, Trimetazidine, Trolnitrate Phosphate and Visnadine;

Vasodilators (peripheral) such as Aluminum Nicotinate, Bamethan,Bencyclane, Betahistine, Bradykinin, Brovincamine, Bufoniode,Buflomedil, Butalamine, Cetiedil, Ciclonicate, Cinepazide, Cinnarizine,Cyclandelate, Diisopropylamine Dichloracetate, Eledoisin, Fenoxidil,Flunarisine, Heronicate, Ifenprodil, Inositol Niacinate, lsoxsuprine,Kallidin, Kallikrein, Moxisylyte, Nafronyl, Nicametate, Nicergoline,Nicofuranose, Nicotinyl Alcohol, Nylidrin, Pentifylline, Pentoxifylline,Piribedil, Protaglandin E1, Suloctidil and Xanthinal Niacinate;

Vasoprotectants such as Benzarone, Bioflavonoids, Chromocarb,Clobeoside, Diosmin, Dobesilate Calcium, Escin, Rolescutol,Leucocyanidin, Metescufylline, Quercetin, Rutin and Troxerutin;

Vitamins, vitamin sources, and vitamin extracts such as Vitamins A, B,C, D, E, and K and derivatives thereof, Calciferols, Glycyrrhiza andMecobalamin;

Vulnerary agents such as Acetylcysteine, Allantoin, Asiaticoside,Cadexomer Iodine, Chitin, Dextranomer and Oxaceprol;

Anticoagulants such as heparin;

Miscellaneous such as Erythropoietin (Hematinic), Filgrastim,Finasterlde (Benign Prostate Hypertrophy) and Interferon β1-α (MultipleSclerosis).

In certain embodiments, the agent to be delivered is one or moreproteins, hormones, vitamins or minerals. In certain embodiments, theagent to be delivered is selected from insulin, IGF-1, testosterone,vinpocetin, hexarelin, GHRP-6 or calcium. In certain embodiments, thecompositions contain two or more agents.

The above list of active agents is based upon those categories andspecies of drugs set forth on pages THER-1 to THER-28 of The MerckIndex, 12th Edition, Merck & Co. Rahway, N.J. (1996). This reference isincorporated by reference herein in its entirety.

D. Uses of the Compositions

Therapeutic and diagnostic applications of the microspheres include drugdelivery, vaccination, gene therapy, and in vivo tissue or tumorimaging. Routes of administration include oral or parenteraladministration; mucosal administration; ophthalmic administration;intravenous, subcutaneous, intra articular, or intramuscular injection;inhalation administration; and topical administration.

The diseases and disorders can include, but are not limited to neuraldisorders, respiratory disorders, immune system disorders, musculardisorders, reproductive disorders, gastrointestinal disorders, pulmonarydisorders, digestive disorders, metabolic disorders, cardiovasculardisorders, renal disorders, proliferative disorders, cancerous diseasesand inflammation.

The microparticles provided herein can be used to treat Infectiousdiseases, such as arboviral infections, botulism, brucellosis,candidiasis, campylobacteriosis, chickenpox, chlamydia, cholera,coronovirus infections, staphylococcus infections, coxsackie virusinfections, Creutzfeldt-Jakob disease, cryptosporidiosis, cyclosporainfection, cytomegalovirus infections, Epstein-Barr virus infection,dengue fever, diphtheria, ear infections, encephalitis, influenza virusinfections, parainfluenza virus infections giardiasis, gonorrhea,Haemophilus influenzae infections, hantavirus infections, viralhepatitis, herpes simplex virus infections, HIV/AIDS, helicobacterinfection, human papillomavirus (HPV) infections, infectiousmononucleosis, legionellosis, leprosy, leptospirosis, listeriosis, lymedisease, lymphocytic choriomeningitis, malaria, measles, marburghemorrhagic fever, meningitis, monkeypox, mumps, mycobacteria infection,mycoplasma infection, norwalk virus infection, pertussis, pinworminfection, pneumococcal disease, Streptococcus pneumonia infection,Mycoplasma pneumoniae infection, Moraxella catarrhalis infection,Pseudomonas aeruginosa infection, rotavirus infection, psittacosis,rabies, respiratory syncytial virus infection (RSV), ringworm, rockymountain spotted fever, rubella, salmonellosis, SARS, scabies, sexuallytransmitted diseases, shigellosis, shingles, sporotrichosis,streptococcal infections, syphilis, tetanus, trichinosis, tuberculosis,tularemia, typhoid fever, viral meningitis, bacterial meningitis, westnile virus infection, yellow fever, yersiniosis zoonoses, and any otherinfectious respiratory, pulmonary, dermatological, gastrointestinal andurinary tract diseases.

Other diseases and conditions, including arthritis, asthma, allergicconditions, Alzheimer's disease, cancers, cardiovascular disease,multiple sclerosis (MS), Parkinson's disease, cystic fibrosis (CF),diabetes, non-viral hepatitis, hemophilia, bleeding disorders, blooddisorders, genetic disorders, hormonal disorders, kidney disease, liverdisease, neurological disorders, metabolic diseases, skin conditions,thyroid disease, osteoporosis, obesity, stroke, anemia, inflammatorydiseases and autoimmune diseases.

E. Combinations, Kits, Articles of Manufacture

Combinations and kits containing the combinations providede hereinincluding microparticles or ingredients for forming the microparticlessuch as a protein or other macromolecule, counterions, solvents,buffers, or salts and optionally including instructions foradministration are provided. The combinations include, for example, thecompositions as provided herein and reagents or solutions for dilutingthe compositions to a desired concentration for administration to a hostsubject, including human beings. The combinations also can include thecompositions as provided herein and additional nutritional and/ortherapeutic agents, including drugs, as provided herein.

Additionally provided herein are kits containing the above-describedCombinations and optionally instructions for administration by oral,subcutaneous, transdermal, intravenous, intramuscular, ophthalmic orother routes, depending on the protein and optional additional agent(s)to be delivered.

The compositions provided herein can be packaged as articles ofmanufacture containing packaging material, a composition providedherein, and a label that indicates that the composition, e.g., a DAS181formulation, is formulated for oral, pulmonary or other delivery.

The articles of manufacture provided herein can contain packagingmaterials. Packaging materials for use in packaging pharmaceuticalproducts are well known to those of skill in the art. See, e.g., U.S.Pat. Nos. 5,323,907, 5,052,558 and 5,033,252. Examples of pharmaceuticalpackaging materials include, but are not limited to, blister packs,bottles, tubes, inhalers, pumps, bags, vials, containers, bottles, andany packaging material suitable for a selected formulation and intendedmode of administration and treatment.

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1 Preparation of Microspheres of the Sialidase Fusion Protein,DAS181

A. Purification of DAS181

DAS181 is a fusion protein containing the heparin (glysosamino glycan,or GAG) binding domain from human amphiregulin fused via its N-terminusto the C-terminus of a catalytic domain of Actinomyces Viscosus(sequence of amino acids set forth in SEQ ID NO:17). The DAS181 proteinwas purified as described in Malakhov et al., Antimicrob. AgentsChemother., 1470-1479, 2006, which is incorporated in its entirety byreference herein. Briefly, the DNA fragment coding for DAS181 was clonedinto the plasmid vector pTrc99a (Pharmacia; SEQ ID NO:16) under thecontrol of a IPTG (isopropyl-R-D-thiogalactopyranoside)-induciblepromoter. The resulting construct was expressed in the BL21 strain ofEscherichia Coli (E. Coli).

The E. Coli cells containing the expressed construct were lysed bysonication in 50 mM phosphate buffer, pH 8.0; 0.3 M NaCl and 10%glycerol. The clarified lysate was passed through an SP-Sepharosecolumn. Proteins were eluted from the column with lysis buffer thatcontained 0.8 M NaCl. The fraction eluted from SP-Sepharose was adjustedto 1.9 M ammonium sulfate ((NH4)₂SO₄), clarified by centrifugation, andloaded onto a butyl-Sepharose column. The column was washed with twovolumes of 1.3 M (NH4)₂SO₄, and the DAS181 fusion protein was elutedwith 0.65 M (NH4)₂SO₄.

For the final step, size exclusion chromatography was performed onSephacryl S-200 equilibrated with phosphate-buffered saline (PBS). Theprotein purity was determined to be greater than 98% as assessed bysodium dodecyl sulfate-polyacrylamide gel electrophoresis,reversed-phase high-pressure liquid chromatography, and enzyme-linkedimmunosorbent assay with antibodies generated against E. Coli cellproteins. The purified DAS181, molecular weight 44,800 Da, was dialyzedagainst 2 mM sodium acetate buffer, pH 5.0.

B. Activity of DAS181

The sialidase activity of DAS181 was measured using the fluorogenicsubstrate 4-methylumbelliferyl-N-acetyl-α-D-neuraminic acid (4-MU-NANA;Sigma). One unit of sialidase is defined as the amount of enzyme thatreleases 10 nmol of MU from 4-MU-NANA in 10 minutes at 37° C. (50 mMCH3COOH—NaOH buffer, pH 5.5) in a reaction that contains 20 nmol of4-MU-NANA in a 0.2 ml volume (Potier et al., Anal. Biochem., 94:287-296,1979). The specific activity of DAS181 was determined to be 1,300 U/mgprotein (0.77 μg DAS181 protein per unit of activity).

C. Preparation of Microspheres Using Purified DAS181

DAS181 (10 mg/ml), purified and prepared as described under Section Aabove, was used to form 200 μl cocktails as shown below. The cocktailscontained either glycine or citrate as counterions, and isopropanol asorganic solvent, as follows:

1) DAS181+5 mM glycine, pH 5.0;

2) DAS181+5 mM glycine, pH 5.0+10% isopropanol;

3) DAS181+5 mM sodium citrate, pH 5.0;

4) DAS181+5 mM sodium citrate, pH 5.0+10% isopropanol;

Plastic microcentrifuge tubes containing the cocktails with ingredientsas described in 1)-4) above were gradually cooled from:

(a) ambient temperature (about 25° C.) to 4° C. by placing the cocktailsin a refrigerator, followed by:

(b) cooling to −20° C. by placing the resulting cocktail from (a) in afreezer, followed by:

(c) freezing to −80° C. by placing the resulting cocktail from b) in afreezer.

Under optimal conditions, microspheres would be expected to form betweenabout 4° C. to about −20° C. (generally in the range of about −2° C. toabout −15° C.). Freezing to −80° C. is carried out to remove ingredientsfrom the cocktail other than the microspheres (e.g., solvent, etc.) byfreeze-drying. Cocktail 4) was prepared in triplicate, two aliquots inplastic tubes and one in a glass tube. One aliquot (in a plastic tube)was cooled as described above, while the two other aliquots (one in aplastic tube and one in a glass tube) were subjected to snapcooling/freezing by dipping the tubes into liquid nitrogen.

Upon freezing, all tubes were placed into the lyophilizer and thevolatiles (water and isopropanol) were removed by sublimation, leavingthe dry pellets.

Results:

The dry pellets recovered from the cocktails treated as described above,were tested for the presence of microspheres. Of the above samples,microspheres with good dispersivity characteristics, about 2 microns(μm) in size, were observed only with cocktail 4) containing citratecounterion and isopropanol and subjected to gradual cooling. Thecounterion glycine did not prove to be optimal for the DAS181 protein(cocktail 2)), showing a mixture of glass-like crystals and agglomerateswith only a few microspheres. When no organic solvent was present, aglass-like mass of lyophilized DAS181 protein was obtained and nomicrospheres were observed (cocktails 1) and 3)). Snap-freezing ofcocktail 4) in a glass tube produced glass-like crystals and nomicrospheres, while snap-freezing of cocktail 4) in a plastic tube(cooling rate is slightly slower due to slower diffusion of heat throughplastic than through glass) produced agglomerated microspheres.

This example demonstrates that microspheres with narrow sizedistribution and good dispersivity (minimal agglomeration) can beproduced by a combination of appropriate protein, counterion, organicsolvent and gradual cooling, using the methods provided herein.

Example 2 Size of DAS181 Microspheres as a Function of Organic SolventConcentration

DAS181 was purified and used to prepare microspheres as described abovein Example 1 (see cocktail 4)), using a combination of DAS181 protein(10 mg/ml), citrate counterion (sodium citrate, 5 mM) and isopropanolorganic solvent (10%, 20% or 30%). The resulting cocktail solutions werecooled from ambient temperature (about 25° C.) to 4° C., followed bycooling to −20° C., followed by freezing to −80° C., as described inExample 1. Upon freezing to −80° C., the tubes are placed in alyophilizer and the volatiles (water and isopropanol) were removed bysublimation, leaving the dry powder containing microspheres.

Results:

Microsphere formation was observed with all three concentrations: 10%,20%, or 30%, of the organic solvent isopropanol. The dimensions of themicrospheres however varied, depending on the concentration of theorganic solvent. The sizes of the microspheres as determined bycomparing the particles to a grid on a hemocytometer were estimated tobe 2 microns using 10% isopropanol, 4 microns using 20% isopropanol, and5-6 microns using 30% isopropanol. These results demonstrate that thesize of the microparticles can be engineered as desired using anappropriate concentration of organic solvent.

Example 3 Size of DAS181 Microspheres as a Function of ProteinConcentration

DAS181 was purified and used to prepare microspheres as described abovein Example 1 (see cocktail 4)), using a combination of DAS181 protein (5mg/ml or 10 mg/ml), citrate counterion (sodium citrate, 5 mM) andisopropanol (5% or 20%). The resulting cocktail solutions were cooledfrom ambient temperature (about 25° C.) to 4° C., followed by cooling to−20° C., followed by freezing to −80° C., as described in Example 1.Upon freezing to −80° C., the tubes were placed in a lyophilizer and thevolatiles (water and isopropanol) were removed by sublimation, leavingthe dry powder containing microspheres.

Results:

Microsphere formation was observed with both concentrations of protein(5 mg/ml and 10 mg/ml), and both concentrations of organic solvent (5%or 20%). The dimensions of the microspheres however varied. Cocktailscontaining 5 mg/ml or 10 mg/ml protein and 5% isopropanol producedmicrospheres estimated to be about 1.5 micron in size. The cocktailcontaining 5 mg/ml protein and 20% isopropanol produced microspheres ofan estimated size of about 3 microns, while the cocktail containing 10mg/ml protein and 20% isopropanol produced microspheres of an estimatedsize of about 4 microns. These results demonstrate that the size of themicroparticles can be engineered as desired using an appropriateconcentration of protein, or an appropriate combination of concentrationof organic solvent and concentration of protein.

Example 4 Size of DAS181 Microspheres as a Function of CounterionConcentration

DAS181 was purified and used to prepare microspheres as described abovein Example 1 (see cocktail 4)), using a combination of DAS181 protein(10 mg/ml), citrate counterion (sodium citrate; 2 mM, 3 mM or 6 mM) andisopropanol (20%). The cocktail solutions were mixed in glass vials andcooled from +20° C. to −40° C. at a freeze ramp of 1° C. per minute in aMillrock Lab Series lyophilizer. Volatiles (water and isopropanol) wereremoved by sublimation at 100 mTorr with primary drying at −30° C. for12 hours and secondary drying at 30° C. for 3 hours, leaving the drypowder containing microspheres.

Results:

Microsphere formation was observed at all three tested concentrations ofcitrate counterion. The size of the microspheres increased from 1 micronat 2 mM citrate, to 3 microns at 3 mM citrate, to 5 microns at 6 mMcitrate. Addition of 1 mM sodium acetate or 1 mM sodium chloride to thecocktail containing 2 mM citrate did not affect formation of themicrospheres triggered by the citrate counterion. These resultsdemonstrate that the size of the microparticles can be engineered asdesired using an appropriate concentration of counterion.

Example 5 DAS181 Microspheres Formed in the Presence of Surfactants

The addition of surfactants to macromolecular (e.g., protein)microspheres often can improve characteristics of the microspheres thatrender them suitable for administration to a subject, such asflowability, dispersivity and disposition for a particular route ofadministration, such as intranasal or oral inhalation. To test whethersurfactants can be incorporated into the methods of manufacturingmicrospheres as provided herein, the production of DAS181 microsphereswas undertaken as described in Example 1 above, except that in addition,a surfactant was added to the solution.

To a cocktail solution containing 5 mg/ml DAS181, 5 mM sodium citrate,and 20% isopropanol, was added a surfactant (3.5% w/w lecithin, 0.7% w/wSpan-85® (sorbitan trioleate), or 3.5% w/w oleic acid). The microsphereswere formed by cooling the solutions to 4° C., followed by cooling to−20° C., followed by freezing to −80° C. for lyophilization as describedabove in Example 1. Upon freezing, the tubes were placed into alyophilizer and the volatiles (water and isopropanol) were removed bysublimation, leaving the dry powder containing microspheres.

Results:

The microspheres resulting from treatment of each of the above cocktailsas described above were spread on glass slides using cover slips rubbedin a circular motion. Efficient microsphere formation was observed inall cases. When the samples containing surfactant were compared to thesample containing all the remaining ingredients but no added surfactant,it was noted that the microspheres formed in the presence of surfactanthad improved dispersivity (lesser agglomeration or aggregation).

Example 6 Preparation of Microspheres of Bovine Serum Albumin (BSA) bySelection of Suitable Types and Concentrations of Organic Solvents andCounterions

As described herein, the methods provided herein can empirically beoptimized in high-throughput format to obtain microspheres havingdesired characteristics including size, flowability and dispersivity.The purpose of this experiment was to demonstrate that by varying typesand concentrations of organic solvents and counterions, as well as pH ofthe cocktail, size and quality of microspheres of a protein of interest,in this case bovine serum albumin (BSA), can be adjusted.

Cocktail solutions containing 5 mg/ml of BSA and various organicsolvents and counterions at indicated pH and concentrations (seeTable 1) were placed in a microtiter plate (final volume per well of 0.1ml). Cocktails were cooled from +20° C. to −40° C. at a freeze ramp of1° C. per minute in a Millrock Lab Series lyophilizer. Volatiles wereremoved by sublimation at 100 mTorr, with a primary drying at −30° C.for 12 hours and secondary drying at 30° C. for 3 hours.

Results:

The results are shown in Table 1 below. For the BSA protein,combinations (of counterion and organic solvent, respectively) thatproduced the most uniform microspheres with minimal crystallization oraggregation include:

(1) citrate+isopropanol(2) citrate+acetone(3) itaconic acid+1-propanol(4) glycine+dioxane(5) glycine+1-propanol(6) rubidium+1-propanol(7) perchlorate+1-propanol

TABLE 1 High-throughput screening of BSA microspheres formed underdifferent conditions Counterion pH Organic Solvent Product description 5mM pivalic 4.0 5% Cyclohexanol 0.5-1 micron microspheres with acidoccasional crystals 5 mM pivalic 4.0 5% 1-propanol 0.5-1 micronmicrospheres with acid some aggregates 5 mM pivalic 4.0 5% butyl alcoholAggregated microspheres acid 5 mM pivalic 4.0 5% p-Dioxane Aggregatedmicrospheres acid 5 mM rubidium 9.0 5% Cyclohexanol 0.5-1 micronmicrospheres. chloride Aggregates and occasional crystals 5 mM rubidium9.0 5% 1-propanol 0.5-1 micron microspheres chloride 5 mM rubidium 9.05% butyl alcohol Few microspheres (0.5-1 chloride micron). Mostlyaggregates and crystals 5 mM rubidium 9.0 5% p-Dioxane 1-2 micronsmicrospheres with chloride some aggregates 5 mM sodium 4.0 5%Cyclohexanol 1-2 microns microspheres with bromide some aggregates 5 mMsodium 4.0 5% 1-propanol Few microspheres (0.5-2 bromide micron). Mostlyaggregates and crystals 5 mM sodium 4.0 5% butyl alcohol Fewmicrospheres (0.5-1 bromide micron). Mostly aggregates and crystals 5 mMsodium 4.0 5% p-Dioxane 1-2 microns microspheres with bromide someaggregates 5 mM sodium 4.0 5% Cyclohexanol 0.5-2 microns microsphereswith perchlorate some crystals and aggregates 5 mM sodium 4.0 5%1-propanol 0.5-1 micron microspheres perchlorate 5 mM sodium 4.0 5%butyl alcohol Few 1-2 microns microspheres. perchlorate Mostly crystalsand aggregates 5 mM sodium 4.0 5% p-Dioxane Aggregated microspheresperchlorate 5 mM calcium 4.0 5% Cyclohexanol Few 1-2 micronsmicrospheres, phosphate mostly aggregates 5 mM calcium 4.0 5% 1-propanol1-2 microns microspheres with phosphate some aggregates 5 mM calcium 4.05% butyl alcohol Few 1-2 micron microspheres. phosphate Mostly crystalsand aggregates 5 mM calcium 4.0 5% p-Dioxane Aggregated microspheresphosphate 5 mM 9.0 5% Cyclohexanol 0.5-1 micron microspheres withtriethylamine some crystals and aggregates 5 mM 9.0 5% 1-propanol 1-2micron microspheres with triethylamine some aggregates 5 mM 9.0 5% butylalcohol Few 1-2 micron microspheres. triethylamine Mostly crystals andaggregates 5 mM 9.0 5% p-Dioxane Aggregated microspheres triethylamine 5mM glycine 9.0 5% Cyclohexanol 0.5-1 micron microspheres with somecrystals and aggregates 5 mM glycine 9.0 5% 1-propanol 0.5-2 micronmicrospheres with occasional aggregates 5 mM glycine 9.0 5% butylalcohol Few 1-2 micron microspheres. Mostly crystals and aggregates 5 mMglycine 9.0 5% p-Dioxane 1-2 micron microspheres 5 mM sodium 4.0 15%isopropanol 1-2 micron microspheres citrate 5 mM sodium 4.0 15% acetone0.5-1 micron microspheres citrate 5 mM 4.0 15% 1-propanol 1-2 micronmicrospheres itaconic acid

These results demonstrate that, for each protein, multiple formulationscan readily be screened for the best microsphere formation (desireddimensions, uniformity, dispersivity, minimal aggregation and crystalformation, etc.) in high-throughput format. The combinations of reagentsand conditions (counterion, organic solvent, pH, concentrations)selected from the initial screen can then further be fine-tuned asdesired.

Example 7 Preparation of Microspheres Using a Variety of Proteins

The methods provided herein can be used to prepare microspheres using avariety of proteins. In addition to DAS181 and BSA exemplified above,the methods were used to prepare microspheres from trypsin, hemoglobin,DNase I, lysozyme, ovalbumin, RNAse A, hexahistidine-tagged humanproteinase inhibitor 8 (PI8, having the sequence of amino acids setforth in SEQ ID NO:15), red fluorescent protein (RFP) and greenfluorescent protein (GFP).

DNase 1, trypsin and hemoglobin were purchased from Worthington.Lysozyme, ovalbumin, and RNAse A were purchased from Sigma. Purificationof 6×His tagged PI8, GFP and RFP: 6×His tagged PI8, GFP and RFP wereexpressed and purified essentially as described for DAS181 in Example 1above, with the following modifications:

Purification of 6×His Tagged GFP and 6×His Tagged RFP:

Constructs encoding Red Fluorescent protein and Green Fluorescentprotein with N-terminal Hiss tags were expressed in E. Coli as6×His-tagged proteins. Expression of Red Fluorescent protein was allowedto proceed overnight in LB medium with 1 mM IPTG. Green Fluorescentprotein was induced for 3 hour in TB medium with 1 mM IPTG. Cell lysatesfrom 4 liters of induced cultures were clarified by centrifugation andthe proteins were purified by metal chelate affinity chromatography onFast-Flow Chelating resin (GE Healthcare) charged with Nickel and packedinto C-10 columns (GE Healthcare).

The proteins were further purified by Gel Filtration Chromatography on a0.5 cm×70 cm Sephacryl 200 column equilibrated with phosphate bufferedsaline. The proteins were dialyzed against 2 mM sodium acetate buffer,pH 5.0, and concentrated on a Centriprep (Amicon).

Purification of 6×His taqqed PI8:

A construct encoding PI8 with an N-terminal Hiss tag was expressed in E.Coli as 6×His-tagged PI8. Purification was performed as described for6×His RFP and 6×His GFP above, with the exception that all buffers usedin the various chromatographic purification steps contained 1 mM TCEP(Tris(2-carboxyethyl)phosphine hydrochloride).

Preparation of Microspheres:

Cocktail solutions containing 5 mg/ml of protein and variouscounterions, organic solvents and pH as listed below were prepared in amicrotiter plate as described above in Example 6.

TABLE 2 Combinations Used to Produce Microspheres of Different ProteinsMicrosphere Organic Size Protein Counterion pH Solvent (microns) Trypsin5 mM 8.0 5% isopropanol 0.5-1   arginine Lysozyme 5 mM citrate 8.0 5%isopropanol 4-5 PIN 168 (PI8) 5 mM citrate 5.0 7% isopropanol 2-5 DNaseI 5 mM citrate 4.0 5% isopropanol 0.4-1   RNase A 5 mM citrate 4.0 5%isopropanol 0.4-1   Hemoglobin 5 mM 5.0 10% 0.4-0.7 glycine isopropanolOvalbumin 5 mM pivalic 4.0 10% 0.5-1   acid isopropanol Red fluorescent5 mM pivalic 7.0 10% 1-propanol 1-4 protein acid (occasional aggregates)Green fluorescent 5 mM pivalic 7.0 10% 1-propanol 0.5-1.5 protein acid

The microtiter plate was cooled from +20° C. to −40° C. at a freeze rampof 1° C. per minute in a Millrock Lab Series lyophilizer. Volatiles(water and isopropanol) were removed by sublimation at 100 mTorr withprimary drying at −30° C. for 12 hours and secondary drying at 30° C.for 3 hours, leaving the dry powder containing microspheres.

The dry powders were spread on glass slides and microphotography wasperformed through either 32× or 100× objective. All the combinationslisted in Table 2 above produced microspheres of good quality (uniformsize distribution, dispersivity, with few aggregates and/or crystals).The microspheres varied in size from about 0.4-1 micron (RNAse A, DNAseI) to about 2-5 microns (6×His PI8, lysozyme), depending on the protein.This example demonstrates that the methods provided herein can be usedto produce microspheres from a wide variety of proteins.

Example 8 Aerodynamic Particle Size Distribution of DAS181 Microspheresfor Inhalation: A Comparison of the Method Provided Herein withSpray-Drying

As described herein, the methods provided herein can be used to producemicrospheres in any desired size range, including a range of about 0.5micron to about 6-8 microns for delivery via inhalation.

A. Preparation of Microspheres

To test the aerodynamic particle size distribution of DAS181 dry powder(microspheres) formulated for delivery by inhalation, DAS181microspheres were prepared using two methods as follows:

(a) A DAS181 aqueous solution containing 14 mg/ml DAS181, 5 mM sodiumcitrate, pH 5.0 was spray dried into an air stream at 55° C., to producemicrospheres.(b) Alternately, DAS181 microspheres were produced according to themethods provided herein. To a DAS181 aqueous solution containing 14mg/ml DAS181, 5 mM sodium citrate, pH 5.0, was added 5% isopropanol asorganic solvent. The resulting solution was cooled from +20° C. to −40°C. at a freeze ramp of 1° C. per minute in a Millrock Lab Serieslyophilizer. Volatiles (water and isopropanol) were removed bysublimation at 100 mTorr with primary drying at −30° C. for 12 hours andsecondary drying at 30° C. for 3 hours, leaving the dry powdercontaining microspheres.

B. Aerodynamic Particle Size Distribution of Microspheres

The microspheres prepared as described in Example 8A were tested byAndersen Cascade Impaction. The deposition of pharmaceuticals in therespiratory tract can be predicted by the aerodynamic behavior ofparticles (microspheres) on the stages/collection plates of the cascadeimpactor.

The cascade impaction experiment was performed using DAS181 microspheresprepared by one of the two alternate methods described in section Aabove, i.e., either by spray-drying or by the methods provided herein.The microspheres (10 mg) were loaded into gelatin capsules. The gelatincapsules were placed into a CycloHaler (PharmaChemie) dry powder inhalerand subjected to cascade impaction. An 8-stage, non-viable AndersenCascade Impactor (Thermo Electron, Boston) modified for use at 90 litersper minute of air flow and equipped with a USP throat, induction coneand no preseparator, was used. The collection plates of the impactorrepresenting various areas/stages of deposition post-inhalation(trachea, primary and secondary bronchi, terminal bronchi, alveoli,etc.) were coated with silicon spray to prevent bouncing of themicrospheres. The microspheres from the stages and collection plateswere recovered into a phosphate buffered saline containing 0.1% Tween,and the amount of deposited DAS181 recovered from each stage andcollection plate was quantified by measuring absorbance at 280 nm.

Results:

The geometric size of microspheres produced by the two methods wasassessed by light microscopy and found to be essentially identical(range of 1.5-3.0 microns) for both methods. As shown in Table 3 below,however, the aerodynamic particle size distribution of the twopreparations differs significantly between the two methods. For themicrospheres produced according to a method as provided herein (i.e.,method (b) as set forth in section A above), less than 25% remainedtrapped in the mouth (throat/cone of the impactor assembly), whilegreater than 70% of the microspheres were delivered to the trachea andlungs (with greater than 40% in the terminal bronchi and alveoli). Incomparison, less than 50% of the DAS181 microspheres formed byspray-drying (method (a) as set forth in section A above) was deliveredto the trachea and lungs (less than 20% in the terminal bronchi andalveoli). The results demonstrate that methods provided herein canproduce microspheres for delivery into deep lungs, and that themicrospheres produced by methods provided herein have superiordisagglomeration and flowability properties (provide a higher delivereddose) compared to microspheres produced by a spray-drying method.

TABLE 3 Results of Cascade Impaction Analyses of DAS181 MicrospheresPercent Deposition of DAS181 Component Microspheres of the ExpectedProduced by Andersen Corresponding Deposition in Method (a) MicrospheresCascade Size Cut-Off Respiratory (i.e., Spray Produced by Impactor(microns) Airways Drying) Method (b) Throat + Cone >10 oral cavity 42.916.6 −2 (S + P) 8.0-10 oral cavity 3.7 4.9 −1 (S + P) 6.5-8.0 oropharynx5.9 5.5 −0   5.2-6.5 pharynx 5.8 4.0 1 3.5-5.2 trachea/bronchi 12.5 9.32 2.6-3.5 secondary 11.6 12.6 bronchi 3 1.7-2.6 terminal bronchi 11.024.0 4 1.0-1.7 alveoli 4.5 19.2 5 0.43-1.0  Alveoli 1.4 3.5

Example 9 Large Scale Manufacture of Microspheres

This example demonstrates that the methods provided herein can be scaledfor the manufacture of large quantities of DAS181. The Batch Processdescribed herein is suitable for the manufacture of high quality drypowder microspheres in an amount ranging from, for example, milligramsto about a kilogram and is limited by the capacity of the mixing tankand/or lyophilizer shelf space. An alternative “continuous” processdescribed herein can be used to manufacture amounts ranging from, forexample, hundreds of grams to hundred or more kilograms (100 grams to100 kg and above). Additional advantage of continuous process is abetter control over the chilling of the cocktail.

The large scale manufacture by a batch process or by a continuousprocess can follow, for example, one or more of the steps describedbelow in any combination of steps or specific alternative methods:

-   -   Precipitation of protein into microspheres. This step can be        performed in a batch mode by placing the cocktail solution        containing the desired concentration of protein, organic solvent        and counterion in lyophilization tray(s) and placing the tray(s)        onto lyophilizer shelves. Alternatively, trays can be chilled        and frozen on a chilled platform or other type of equipment        (e.g., a freezer) and stored for a period of time frozen and        lyophilized later. Alternatively, the microspheres can be formed        by precipitation in a vessel with stirring, wherein the vessel        is placed onto a cold surface or a cooling coil is immersed into        liquid or while the cocktail is being recirculated through a        heat exchanger using a peristaltic pump. Alternatively, the        microspheres can be formed by precipitation in a continuous        mode, by passing the cocktail solution through a heat        exchanger(s) once using a peristaltic pump.    -   Removal of bulk liquid. The suspension of the microspheres can        be concentrated using standard centrifugation, continuous flow        centrifugation (e.g., CARR ViaFuge Pilot), or filtration (e.g.,        on glass fiber, sintered glass, polymer filters, hollow fiber        cartridges (e.g., those manufactured by GE Healthcare) or        tangential flow filtration cassettes (TFF cassettes, such as        those manufactured by Millipore or Sartorius)). The removal of        bulk liquid (50% or greater) can result in a faster drying cycle        and higher efficiency and throughput.    -   Drying the microspheres. The recovered microspheres formed by        any mode, can be dried by conventional lyophilization.        Alternatively, the microspheres can be dried under ambient        temperature and atmospheric pressure, eliminating the use of        lyophilizer.

Results:

DAS181 protein was successfully processed into dry powder (microspheres)by a continuous mode as described herein. Cocktail containing 10 mg/mlDAS181, 20% isopropanol, 2 mM sodium sulfate was passed through 35SERIES heat exchanger (Exergy, Garden City, N.Y.) coupled with a NESLABcirculating cryostat using a peristaltic pump so that during the passagethe cocktail was cooled from about 25° C. to about −12° C. The resultingsuspension of microspheres exiting the heat exchanger was pumped into aprechilled lyophilization tray (−40° C.), frozen and lyophilized or,alternatively, pumped directly into liquid nitrogen and thenlyophilized. The resulting microspheres, which were analyzed bymicroscopy and cascade impaction, showed uniform microspheres withminimal aggregation and good dispersivity and were similar in dimensionsand aerodynamic particle size distribution to the microspheres producedby batch mode. When the formulated DAS181 cocktail solution was notchilled (not passed through heat exchanger, thus no precipitation ofmicrospheres was induced) and poured directly into liquid nitrogen, nomicrospheres were observed and, instead, glass-like crystals wereobserved after lyophilization.

Example 10 Batch Mode Process and Formulation of DAS181 Microspheres forDelivery to Upper and Central Respiratory Airways

This example describes formulation and a process for manufacture ofDAS181 microspheres. The contents of the DAS181 cocktail solution andtheir relative amounts are shown in Table 4 below.

TABLE 4 Batch Manufacturing Formula for DAS181 Microspheres. FinalAmount for one batch⁽¹⁾ concentration Stock solution Amount informulated Ingredient concentration added cocktail Function DAS181protein 19.55 g/L 3.306 L, 12 g/L Active ingredient Sodium acetate⁽²⁾1.12 mM API 0.688 mM pH buffer Acetic acid⁽²⁾ 0.63 mM solution 0.0387 mMpH buffer Sodium Sulfate 500 mM 0.0215 L 2 mM Microparticle formationagent (counterion) Isopropanol 100% v/v 0.269 L 5% v/v Microparticleformation agent Calcium chloride 500 mM 0.0028 L 0.268 mM Stabilityenhancing agent Water for neat 1.79 L NA Diluent irrigation ⁽¹⁾Batchsize: final volume of formulated cocktail 5.38 L. Theoretical yield 74 gof bulk DAS181 Dry Powder. ⁽²⁾Components of the DAS181 protein (API)stock solution.

A. Production of Bulk Drug Substance

The terms Drug Substance, Active Pharmaceutical Ingredient, and API areused interchangeably in this example and refer to the DAS181 protein.Production of DAS181 protein in bulk was conducted as follows. First,bulk amounts of DAS181 were expressed in E. coli (BL21 strain)essentially as described in Example 1. The E. coli cells expressing theDAS181 protein were washed by diafiltration in a fermentation harvestwash step using Toyopearl buffer 1, UFP-500-E55 hollow fiber cartridge(GE Healthcare) and a Watson-Marlow peristaltic pump.

The recombinant DAS181 protein was then purified in bulk from the cells.The detailed specifications of the components and buffers used in thebulk purification of DAS181 are provided in Tables 5 and 6 below. Theharvested and washed cells were lysed in a homogenization step bypassing the cells twice through using Niro-Soave Panda cell disruptor.The homogenate thus obtained was clarified by microfiltration using theToyopearl buffer 1, Hydrosart 0.2 micron TFF cassette and a WatsonMarlow pump. The clarified homogenate was then concentrated by allowingthe lysate to recirculate without fresh buffer feed. Next, DAS181protein was captured from the clarified homogenate on a ToyopearlSP-550C resin which was washed in a series of buffers (see Table 5)before the DAS181 protein was eluted from the resin. The sodium chlorideconcentration of the eluate was adjusted to 1.0 M in a final buffer of50 mM phosphate at pH 8.0. The DAS181-containing eluate was then passedthrough a Toyopearl Hexyl-650C resin for further purification using aToyopearl Buffer 4. The resin eluate containing DAS181 protein was thenbuffer-exchanged into 5 mM sodium acetate in a diafiltration step (seestep 8 in Table 5). The concentrated protein was next passed through aSartorius Q SingleSep Filter in order to remove DNA in a flow-throughmode. Isopropanol was added to the Q SingleSep filtrate to a finalconcentration of 20% v/v. The DAS181 protein in the buffer was passedthrough an Amberchrome CG300M resin equilibrated with an Amberchrombuffer (see step 11 in Table 5). The purified bulk DAS181 protein wasthen buffer-exchanged into formulation buffer and concentrated bydiafiltration (see step 12 of Table 5).

TABLE 5 Purification of bulk DAS181 drug substance 1 PurposeFermentation Harvest Wash Cartridge GE UFP-500-E55 SpecificationsActivity Buffer Name Inlet PSI Diafiltration Toyopearl Buffer 1 25-35 2Purpose Homogenization Activity Step Buffer Name EquilibrationEquilibration Harvest Buffer Homogenization 1st Pass Sample LoadHomogenization 2nd Pass Sample Load 3 Purpose Homogenate Clarification(Diafiltration) TFF Cartridge HydroSart 10K 0.6 m² SpecificationsActivity Buffer Name Inlet PSI Recirculation Sample Load   40Diafiltration Toyopearl Buffer 1 <50 4 Purpose Permeate ConcentrationTFF Cartridge HydroSart 10K 0.6 m² Specifications Activity Buffer NameInlet PSI Recirculation Sample Load NS Concentration Sample Load <50 5Purpose DAS181 capture performed in bind and elute mode Resin ToyopearlSP-550C Activity Step Buffer Name Loading Sample Load Clar. HomogenateWash SP Wash 1 Toyopearl Buffer 1 SP Wash 2 Toyopearl Buffer 2 SP Wash 3Toyopearl Buffer 3 SP Wash 4 Toyopearl Buffer 2 SP Wash 5 ToyopearlBuffer 1 Elution Elution Toyopearl Buffer 4 6 Purpose Adjust NaClConcentration Method Add NaCl to 1.0M Final Buffer 50 mM phosphate, 1.0MNaCl, pH 8.0 7 Purpose DAS181 purification in flow-through mode ResinToyopearl Hexyl-650C Activity Step Buffer Name Loading Sample Load Cond.Hexyl Load 8 Purpose Concentration & Diafiltration TFF CartridgeHydroSart 10K 0.6 m² Specifications Activity Buffer Name Recirc. L/min*Recirculation Toyopearl Buffer 6 15-16 Concentration Hexyl Product Pool15-16 Diafiltration Toyopearl Buffer 6 15-16 Recirculation ToyopearlBuffer 6 NS 9 Purpose Remove DNA in flow-through mode Resin Sartorius QSingleSep Filter Activity Step Buffer Name Loading Sample Load 10Purpose Buffer Adjustment Method Add Isopropanol to 20% Final Buffer 5mM Acetate, 20% Isopropanol, pH 5.0 11 Purpose DAS181 polishing inflow-through mode Resin Amberchrome CG300M Activity Step Buffer NameLoading Sample Load Amberchrom Load 12 Purpose Concentration &Diafiltration TFF Cartridge HydroSart 10K 0.6 m² Specifications ActivityBuffer Name Recirc. L/min* Recirculation Formulation Buffer 15-16Concentration Amberchrom Product Pool 15-16 Diafiltration FormulationBuffer 15-16 *Volumes in liters, except 4x denotes multiples of theretentate volume CV = Column Volumes NR = Not Recorded NS = NotSpecified

TABLE 6 Buffers used during the DAS181 purification process Buffer NameBuffer Composition Toyopearl Buffer 1 50 mM potassium phosphate, 0.3MNaCl, pH 8.0 Toyopearl Buffer 2 1.1 mM potassium phosphate, 2.9 mMsodium phosphate, 154 mM NaCl, pH 7.4 Toyopearl Buffer 3 1.1 mMpotassium phosphate, 2.9 mM sodium phosphate, 154 mM NaCl, 1% TritonX-100, 0.1% SDS, 0.5% sodium deoxycholate, pH 7.4 Toyopearl Buffer 4 50mM potassium phosphate, 1.0M NaCl, pH 8.0 Toyopearl Buffer 5 50 mMpotassium phosphate, 0.5M NaCl, pH 8.0 Toyopearl Buffer 6 5 mM sodiumacetate, pH 5.0 Toyopearl Buffer 7 5 mM sodium acetate, 60% isopropanol,pH 5.0 Formulation Buffer 1.75 mM sodium acetate, pH 5.0 3% IsoproylAlcohol 3% isopropanol Amberchrom Buffer 5 mM sodium acetate, 20%isopropanol, pH 5.0 adjusted with acetic acid 1.0N NaOH 3% 1.0N NaOH, 3%isopropanol Isopropanol 1.0N NaOH 1.0N NaOH 0.5N NaOH 0.5N NaOH 0.1NNaOH 0.1N NaOH 70% Isopropyl 70% isopropanol Alcohol 20% EtOH 20% ethylalcohol

B. Batch Manufacturing Process

The ingredients set forth in Table 4 above were combined to form DAS181microspheres in a large scale batch process as described below.

Step I: Thawing of Bulk Drug Substance

Frozen 0.2 μm-filtered bulk Drug Substance in plastic bottles was thawedovernight at ambient temperature (25±3° C.).

Step II: Weighing of the Excipients and Preparation of Solutions

35.51 g of Sodium Sulfate anhydrous powder was weighed and Q.S. to 500mL with Water For Irrigation, then stirred to obtain a clear solution.18.38 g of Calcium Chloride dihydrate powder was weighed and Q.S. to 250mL with Water For Irrigation, then stirred to obtain a clear solution.

Step III: Preparation of the DAS181 Cocktail Solution

To 3.3 L of concentrated Drug Substance (19.55 g/L), 1.79 L of Water ForIrrigation was added slowly with stirring, followed by 0.0215 L ofSodium Sulfate solution, 0.0028 L of Calcium Chloride solution and 0.269L of isopropanol. The solution was stirred to ensure complete mixing ofcomponents.

Step IV: Filtration of Formulated Cocktail Solution Through 0.2 μmFilter

The formulated cocktail solution of Step III was filtered through a 0.2μm filter into sterile media bags to control particulates and bioburden.

Step V: Filling into Lyophilization Trays

The formulated filtered solution was dispensed into autoclaved Lyoguardlyophilization trays. To ensure even cooling of the solution andformation of high quality microspheres, 6 trays were each filled with0.9 L or less of cocktail solution.

Step VI: Freezing and Lyophilization

The trays were placed onto lyophilizer (Hull 120FSX200) shelvespre-chilled to −45±5° C. and the solution was allowed to chill andfreeze. Formation of microspheres occured while the solution was beingfrozen. The freezing is allowed to proceed for 1-2 h to ensure completesolidification. The product temperature was verified by reading thethermocouples attached to two of the six trays.

The lyophilization cycle steps are as follows:

-   -   a) Set vacuum to 160 microns and allow to evacuate to 100-200        microns;    -   b) Ramp shelf temperature to +10° C. over 3 h;    -   c) Hold shelf temperature at +10° C. for 36 h (primary drying);    -   d) Thermocouple traces examined to verify that primary drying        phase is completed and the product temperature has stabilized at        +10° C.±5° C. for 15-30 h.    -   e) Ramp shelf temperature to +30° C. over 1 h and hold for 3-5 h        (secondary drying).        Step VII: Transfer of Bulk DAS181 Microspheres into Container        and Mixing

A section on the bottom film of each Lyoguard lyophilization tray wascleaned using sanitizing wipes and a 3×3 cm opening was made with ascalpel. The dry microspheres were transferred into a plastic bottle.The bottle was capped and tumbled forty times, changing directions witheach inversion. The tumbling was to ensure uniformity of bottle content.Samples for analytical testing were taken and the bottle was recappedand sealed into plastic bags for storage.

In the DAS181 microsphere bulk manufacturing process as described above,sulfate was demonstrated to be a safe substance for use as a counterion,and reproducibly produced microspheres with a narrow size distribution.Further, the organic solvent isopropanol was a good solvent of choicebecause (1) a class 3 solvent, (2) it can produce microspheres in a widerange (2-30%, v/v) of concentrations, and (3) it has a relatively highfreezing point so its vapors can efficiently be trapped duringlyophilization.

The protein concentration in the final formulation could be varied(10-14 mg/ml), as could the concentration of counterion (1-5 mM) andisopropanol (2-30% v/v), without substantial impact on the physicalproperties of the microspheres or the activity of the DAS181 protein inthe microspheres. At higher concentrations of isopropanol (15-30%), themicrospheres formed while the cocktail was still fully liquid. At lowerconcentrations (2-15%), ice crystals began to form first, followed byprecipitation to form microspheres.

C. Yield of DAS181 in the Microspheres

The theoretical yield of DAS181 in the dry microspheres is calculatedaccording to the following formula:

Theoretical yield=DAS181 protein,g÷protein fraction in Dry Powder(microspheres)

The protein fraction value (0.866) was established empirically byanalysis of several manufactured batches of DAS181 microspheres. Thetheoretical yield for the amounts as set forth in Table 2 is 64.56g÷0.866=74.55 g. The actual yield of DAS181 Dry Powder was found to be64 g.

Results:

The suitability of the microspheres prepared as described in section Babove for administration by oral inhalation was tested by AndersenCascade Impaction. The results are summarized in Table 7 below. Thedeposition of pharmaceuticals in the respiratory tract can be predictedby deposition of particles (microspheres) on the stages/collectionplates of the cascade impactor. For a pharmaceutical, e.g., DAS181microspheres, that is administered to prevent or treat viral infectionsthat initiate in the respiratory tract, such as influenza, it isdesirable to deposit the pharmaceutical in the throat, trachea, bronchi(upper and central respiratory airways). The DAS181 fusion proteindelivered to upper and central respiratory airways cleaves off thereceptor sialic acids from mucous membranes, thus preventing viralbinding and infection at these sites. For optimal delivery of the DAS181microspheres to sites where respiratory viral infection can beinitiated, i.e., in the throat, trachea or bronchi, the microspheresmust not be (a) so big that they are trapped at the front end in themouth (i.e., microspheres are too big, about 8 microns or greater); or(b) so small that they are deposited in deep lungs and absorbedsystemically into the blood stream (i.e., 0.5 microns or smaller). Fordelivery of the DAS181 microspheres to the throat, trachea and bronchi,a size range of about 1 micron to about 5.5-6 microns generally issuitable.

DAS181 microspheres manufactured as described above were characterizedby Andersen cascade impaction and found to be suitable for delivery toupper and central respiratory airways with sufficiently low percentage(<5%) deposited in the alveoli.

TABLE 7 Aerodynamic Particle Size Distribution of DAS181 dry powder at60 liters per minute. Percent of DAS181 total Component of proteinDAS181 Andersen Cascade Corresponding size Expected deposition indeposited (in protein Impactor cut-off, microns respiratory airways mg)recovered Inhaler (Cyclohaler) 1.57 ± 0.11 20.13% Throat/Cone >10 Oralcavity 0.93 ± 0.19 11.92% −1 (Stage + Plate) 8.6-10  Oral cavity 0.50 ±0.10  6.41% −0 (Stage + Plate) 6.5-8.6 oropharynx 0.40 ± 0.03  5.13%   1(Stage + Plate) 4.4-6.5 pharynx 0.58 ± 0.03  7.44%   2 (Stage + Plate)3.3-4.4 trachea/bronchi 0.83 ± 0.07 10.64%   3 (Stage + Plate) 2.0-3.3Secondary bronchi 1.80 ± 0.09 23.08%   4 (Stage + Plate) 1.1-2.0Terminal bronchi 0.82 ± 0.08 10.51%   5 (Stage + Plate) 0.54-1.1 alveoli 0.23 ± 0.03  2.95%   6 (Stage + Plate) 0.25-0.54 alveoli 0.14 ±0.03  1.79% ΣACI (Emitted) 6.24 ± 0.10 80.00% 10 ± 1.0 mg of DAS181 DryPowder (8.5 mg ± 10% DAS181 protein) was filled into HPMC capsule ΣACI(Emitted) fraction is the sum of all material recovered from USP Throat,Induction Cone and stages −1 to 6.DAS181 microspheres were further characterized by laser diffraction,which demonstrated, consistent with the cascade impaction results, thatthe majority of the microspheres produced by the method described inthis Example are within a size range of between 1 micron and 5 micronsin size. Scanning Electron Microscopy (FEI Quanta 200 Scanning ElectronMicroscope, Everhart Thornley (ET) detector) of the DAS181 microspheresprepared according to the method described in this Example revealed thatthe microspheres are present as agglomerates of hundreds and thousandsof individual particles approximately 0.5-3 micron in size. Theagglomerates however are easily dissipated by air turbulence producedduring the actuation through dry powder inhaler (as demonstrated byAndersen Cascade Impaction or laser diffraction). Light microscopy ofmicrospheres dispersed in a liquid surfactant (e.g. Triton X-100 orTween 20) or non-polar solvent (e.g., alcohol, acetone, or acetonitrile)that does not dissolve the microspheres, confirmed that aggregates areeasily dissipated into individual uniform microspheres.

Example 11 Preparation of DAS181 Microspheres Using Sulfates Other thanthe Sodium Salt

Studies have shown that in certain instances, e.g., in some asthmatics,the presence of sodium in formulations for pulmonary administrationcould carry a risk of inducing airway hyperresponsiveness (Agrawal etal., Lung, 183:375-387 (2005)). This example therefore tested alternatesalts, such as salts of other metals such as potassium, magnesium andcalcium.

DAS181 microspheres were manufactured as described above in Example 1.Cocktail solutions containing 12 mg/mL DAS181 and 5% (v/v) isopropanolcontained as counterions the indicated sulfates at 2 mM concentration,pH 4.5-5.0. The microspheres were formed by cooling the solutions from+25° C. to −45° C. Upon freezing, the volatiles (water and isopropanol)were removed by sublimation, leaving the dry powder containingmicrospheres.

The aerodynamic particle size distribution of the dry powder wasassessed by Andersen Cascade Impaction, and the amount of DAS181 perstage was determined by UV measurement at 226 nm (A₂₂₆). The results areshown below in Table 8. The results demonstrate that sulfate salts otherthan the sodium salt can be used as counterion to obtain DAS181microspheres of a size range such that the majority are delivered to thethroat, trachea and bronchi, in an amount that is comparable to theamount delivered when sodium sulfate is used as the counterion.

TABLE 8 Aerodynamic Particle Size Distribution of DAS181 microspheresformulated with or without sodium Expected Corresponding deposition inPercent DAS181 per stage size cut-off, respiratory Sodium PotassiumMagnesium Calcium microns airways Sulfate Sulfate Sulfate SulfateInhaler 19.86% 28.58% 21.41% 16.71% Capsule 2.07% 2.30% 1.88% 0.00%Throat + Cone >10 Oral cavity 11.67% 9.00% 12.91% 16.79% −1 (S + P) 8.6-10  Oral cavity 10.00% 3.43% 7.86% 14.87% −0 (S + P)  6.5-8.6oropharynx 5.30% 3.08% 4.71% 7.77% 1 (S + P) 4.4-6.5 pharynx 6.97% 5.86%6.58% 7.54% 2 (S + P) 3.3-4.4 trachea/bronchi 7.55% 8.24% 6.90% 6.43% 3(S + P) 2.0-3.3 Secondary 19.57% 20.21% 17.01% 12.65% bronchi 4 (S + P)1.1-2.0 Terminal bronchi 12.39% 14.00% 13.00% 10.39% 5 (S + P) 0.54-1.1 alveoli 2.80% 2.99% 4.31% 4.69% 6 (S + P) 0.25-0.54 alveoli 1.82% 2.31%3.44% 2.16%

The dry powders also were incubated at +37° C. or +53° C. for a durationas indicated in Table 9 and tested for sialidase activity using the4-MU-NANA assay as described in Example 1 and incorporated by referenceherein. The relative activity compared to non-lyophilized DAS181microspheres stored at −80° C. is shown in Table 9. The results showthat the stability of the microspheres prepared using the various metalsulfates as counterions were comparable to that of sodium sulfate, withretention of almost all or all the activity for over 2 months at 37° C.and retention of almost all (sodium and potassium sulfates) or over 85%(magnesium and zinc sulfates) of the activity for over 10 days at 53° C.This experiment demonstrates that various non-sodium containingcounterions can produce microspheres with desirable characteristics.

TABLE 9 Sialidase activity of DAS181 microsphere formulations:accelerated stability studies. Percent Activity Remaining Temperature37° C. 53° C. Incubation Days 42 Days 69 Days 11 Days 39 Days 2 mMSodium Sulfate + 107.14% 105.62% 110.66% 23.66% 0.268 mM CaCl₂ 2 mMPotassium Sulfate +  97.37% 104.00% 101.54% 52.76% 0.268 mM CaCl₂ 2 mMMagnesium 123.81% 107.29%  85.93% 60.00% Sulfate + 0.268 mM CaCl₂ 13.34mM Calcium/2 mM 116.67%  93.20%  87.12% 40.48% Sulfate

Example 12 Stability of DAS181 Microspheres

The stability of the DAS181 protein in the microspheres was assessed bymeasuring sialidase activity over time using the 4-MU-NANA activityassay as described above in Example 1 and as incorporated by referenceherein. The production of dry DAS181 microspheres was undertaken in acocktail solution containing 10 mg/mL DAS181, 2 mM sodium sulfate, 5%v/v isopropanol. To some solutions, 0.01% w/v sugar (sorbitol, mannitol,trehalose or sucrose) was added. The microspheres were formed by coolingthe solutions from +25° C. to −45° C. Upon freezing, the volatiles(water and isopropanol) were removed by sublimation, leaving the drypowders containing microspheres.

A. Stability of DAS181 Microspheres without Sugars

The DAS181 dry powder microspheres formulated without sugars were storedat room temperature (25° C.) in a container next to Drierite desiccant(Hammond Drierite, Xenia, Ohio). The dry powder retained its originalpotency (as measured by sialidase activity using 4-MU-NANA according toExamplel and as incorporated by reference herein; results shown in Table10) and aerodynamic particle size distribution (as measured by AndersenCascade impaction; Table 11) for at least 8 months.

TABLE 10 Specific activity of DAS181 dry powder. Test Time 0 3 months 8months Sialidase Activity with 100% 102.0% 99.9% reference to time 0

TABLE 11 Aerodynamic particle size distribution of DAS181 dry powderExpected Corresponding deposition in ACI size cut-off, respiratoryComponent microns airways Time 0 3 Months 8 Months Throat + >10 Oralcavity 19.57 ± 2.43 26.00 ± 0.30 18.57 ± 4.14  Cone Stage −1 8.6-10 Oral cavity 17.87 ± 0.51 12.87 ± 1.56 15.13 ± 2.41  Stage −0 6.5-8.6oropharynx 10.27 ± 0.93  7.07 ± 0.32 9.80 ± 1.80 Stage 1 4.4-6.5 pharynx 8.57 ± 0.49  8.80 ± 0.26 7.73 ± 0.57 Stage 2 3.3-4.4 trachea/bronchi10.67 ± 0.23 10.70 ± 0.35 9.30 ± 0.82 Stage 3 2.0-3.3 Secondary 21.10 ±0.75 21.80 ± 0.52 21.90 ± 0.87  bronchi Stage 4 1.1-2.0 Terminal 10.10 ±0.75 10.63 ± 0.80 14.50 ± 3.22  bronchi Stage 5 0.54-1.1  alveoli  1.47± 0.23  1.73 ± 0.06 2.37 ± 0.06 Stage 6 0.25-0.54 alveoli  0.33 ± 0.06 0.40 ± 0.10 0.73 ± 0.06 Table 11: Aerodynamic particle distribution wasassessed by Andersen Cascade Impaction and expressed as % of totalDAS181 protein recovered. Capsules were filled with 10 mg of DAS181 drypowder and actuated using Cyclohaler dry powder inhaler as deliverydevice. Air flow rate was 60 Liters per minute. Assays were performed intriplicate, mean and standard deviation are shown.

B. Stability of DAS181 Microspheres Formulated with Sugars

The sialidase activity of DAS181 in the dry powder microsphereformulations containing sugars and in the unlyophilized microsphereformulations stored at −80° C., were measured using fluorescentsubstrate 4-MU-NANA as described in Example 1 and as incorporated byreference herein. The dry powder formulations containing no sugar orvarious sugars as indicated below in Table 12 were stored at +42° C. for4 weeks (forced degradation). The results are shown in Table 12.Relative to unlyophilized formulations stored at −80° C., theformulation containing no sugar retained almost 80% of its activity. Theaddition of various sugars increase the stability so that about 88-98%of the activity is retained, depending on the sugar.

TABLE 12 Percent Sialidase Activity Sugar Remaining after 4 weeks at 42°C. No Sugar 79.82 Sorbitol 91.23 Mannitol 89.47 Trehalose 97.37 Sucrose88.60

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

1. A method of making a protein-based composition, comprising: a) addinga counterion to a solution containing the protein in an aqueous solvent;b) adding an organic solvent to the solution; and c) gradually coolingthe solution to a temperature below about 25° C., whereby a compositioncontaining microparticles comprising the protein is formed, whereinsteps a), b) and c) are performed simultaneously, sequentially,intermittently, or in any order. 2.-141. (canceled)